WO2023153496A1 - Eosinophilia or bronchial asthma biomarker - Google Patents

Eosinophilia or bronchial asthma biomarker Download PDF

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WO2023153496A1
WO2023153496A1 PCT/JP2023/004548 JP2023004548W WO2023153496A1 WO 2023153496 A1 WO2023153496 A1 WO 2023153496A1 JP 2023004548 W JP2023004548 W JP 2023004548W WO 2023153496 A1 WO2023153496 A1 WO 2023153496A1
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protein
domain
group
immunoglobulin
receptor
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PCT/JP2023/004548
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Japanese (ja)
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吉人 武田
華子 吉村
淳 熊ノ郷
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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
    • 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

Definitions

  • the present invention relates to eosinophilia or bronchial asthma biomarkers and the like.
  • Bronchial asthma is defined as "a disease characterized by clinical symptoms such as fluctuating airway narrowing and coughing, characterized by chronic inflammation of the airways.” According to a recent survey, the total number of asthma patients receiving continuous medical care in Japan is more than 1 million, and the total number of asthma patients in the world is about 300 million. There are no clear diagnostic criteria for asthma, and guidelines for asthma diagnosis are defined in the Asthma Prevention and Management Guidelines. In addition to repeated paroxysmal dyspnea and coughing, it is recommended to refer to the presence of reversible airflow limitation, increased airway hyperresponsiveness, presence of atopic predisposition, and presence of airway inflammation. It is necessary to make a diagnosis by comprehensively judging the indicators. Biomarkers currently used to diagnose asthma include blood and sputum eosinophil counts, exhaled nitric oxide (FeNO), and serum IgE levels. Have difficulty.
  • proteomics which is the omics closest to the phenotype, is of particular interest as a biomarker.
  • proteins cannot be amplified, and a comprehensive analysis system has not been established. Therefore, proteomics research for diseases is currently lagging behind.
  • Serum is considered an ideal test sample because it can be collected non-invasively and repeatedly. Therefore, from the viewpoint of proteomics, its usefulness as test data is low.
  • a recent paper on biomarkers that can distinguish between COPD and asthma attempted to analyze dozens of patients, but the analysis was based on an old mass spectrometry (MS) technique based on two-dimensional electrophoresis. The biomarkers found were not only
  • An object of the present invention is to provide a biomarker for eosinophilia or bronchial asthma and a method for using the same.
  • the present inventors found that extracellular vesicles in body fluids collected from subjects or specific protein groups in body fluids are useful as biomarkers for eosinophilia or bronchial asthma. I found As a result of further research based on this knowledge, the present invention was completed. That is, the present invention includes the following aspects.
  • Section 1 A method of testing for eosinophilia or bronchial asthma, comprising: (1) Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D), protein in extracellular vesicles of body fluid collected from a subject or body fluid Group (E), Protein Group (F), and Protein Group (G): (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49, (B)
  • Section 4 The examination method according to any one of Items 1 to 3, wherein the disease to be examined is eosinophilic asthma.
  • Item 5 wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (F) and protein group (G).
  • Item 6 When the detected protein contains at least one protein (protein F') selected from the protein group (F), (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value;
  • the inspection method according to Item 4 or 5, comprising:
  • Item 7 When the detected protein contains at least one protein (protein G') selected from the protein group (G), (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; 7.
  • the inspection method according to any one of items 4 to 6.
  • Item 8 The examination method according to any one of items 1 to 3, wherein the disease to be examined is atopic asthma.
  • Item 10 When the detected protein contains at least one protein (protein D') selected from the protein group (D), (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value;
  • the inspection method according to Item 8 or 9, comprising:
  • Item 11 When the detected protein contains at least one protein (protein E') selected from protein group (E), (2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value;
  • Item 12 The inspection method according to any one of items 1 to 11, wherein the proteins to be detected include protein group (A).
  • the detected protein contains at least one protein (protein A') selected from protein group (A), (2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future;
  • the inspection method according to any one of Items 1 to 12, comprising:
  • biomarkers for eosinophilia or bronchial asthma can be provided.
  • the biomarker it is possible to test for eosinophilia or bronchial asthma, prevent or treat eosinophilia or bronchial asthma, and use a preventive or therapeutic agent for eosinophilia or bronchial asthma. Screening of active ingredients, evaluation of inducing or aggravating eosinophilia or bronchial asthma, and the like may become possible.
  • FIG. 1 shows the measurement results of Galectin-10 expression levels in extracellular vesicle fractions (Exosomes) in Test Example 6.
  • FIG. 1 shows the measurement results of Galectin-10 expression level in tissues (inferior nasal turbinate and polyp) of Test Example 6.
  • FIG. Fig. 8 shows measurement results of Galectin-10 amounts in exosomes and cell lysates with and without eosinophil activation treatment (IL-5+PAF (+)/(-)) in Test Example 8.
  • IL-5+PAF (+)/(-) shows the measurement results of proteomics analysis in Test Example 2.
  • FIG. Each graph information shows the protein to be measured. The vertical axis indicates the expression level in serum exosomes.
  • HC indicates healthy subjects
  • ABA indicates atopic asthmatic subjects
  • EBA indicates eosinophilic asthmatic subjects.
  • 2 shows the results of immunoblotting in Test Example 9.
  • FIG. HC indicates healthy subjects
  • COPD indicates COPD subjects
  • BA indicates asthmatic subjects.
  • 2 shows the results of fluorescent immunostaining in Test Example 10.
  • FIG. HC indicates healthy subjects and BA indicates asthmatic subjects.
  • 2 shows the proteomics results of Test Example 11.
  • FIG. HC indicates healthy subjects and BA indicates asthmatic subjects.
  • a method for measuring and calculating the degree of bronchial wall thickening (WA%) is shown.
  • FIG. 12 shows the correlation between the expression level of Galectin-10 in serum exosomes in Test Example 12, the blood eosinophil count, and the degree of bronchial wall thickening (WA%).
  • the expression level of Galectin-10 in serum exosomes of Test Example 12 and the correlation between blood eosinophil count and respiratory function (FEV1%) are shown.
  • FIG. 10 shows the correlation coefficient (horizontal axis) between the extracellular vesicle fraction and the tissue expression level in Test Example 6.
  • a method for testing eosinophilia or bronchial asthma comprising: (1) cells of a body fluid collected from a subject; From protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G) in outer vesicles or body fluids
  • the present invention relates to an inspection method (also referred to herein as the "inspection method of the present invention") comprising the step of detecting at least one protein selected from the group consisting of: This will be explained below.
  • Process (1) The type of "eosinophilia", which is the disease to be examined, is not particularly limited. All classes, grades, and stages of eosinophilia in various classification criteria for progression of eosinophilia can be tested. Specific examples of eosinophilia include allergic diseases, skin diseases, infectious diseases, respiratory diseases, granulomatous diseases, digestive diseases, endocrine diseases, autoimmune diseases, malignant tumors, and the like. mentioned.
  • allergic diseases include bronchial asthma, atopic dermatitis, urticaria, allergic rhinitis, drug allergy, allergic rhinitis/conjunctivitis, allergic bronchi, allergic granulomatous vasculitis, angioedema/Quincke's edema, Oral mucosal allergy syndrome, allergic purpura, and the like.
  • Skin diseases include, for example, pemphigus vulgaris, eosinophilic pimples, folliculitis, eczema, eosinophilic fasciitis, drug eruption, psoriasis, lymphomatous papules, and erythroderma.
  • Infectious diseases include, for example, parasitism, pulmonary aspergillosis, tuberculosis, syphilis, toxoplasmosis, visceral tick disease, scarlet fever, primary immunodeficiency syndrome, and carinii pneumonia.
  • respiratory diseases include bronchial asthma, eosinophilic lung infiltration, pneumoconiosis/silicosis, hypersensitivity pneumonitis, and idiopathic septic iron.
  • granulomatous diseases include eosinophilic granulomatosis, Wegener's granulomatosis, sarcoidosis, and Kimura disease.
  • Gastrointestinal diseases include, for example, eosinophilic gastroenteritis, allergic gastroenteritis, ulcerative colitis, pancreatitis, and inflammatory bowel disease.
  • Endocrine diseases include, for example, Addison's disease and hyperthyroidism.
  • Autoimmune diseases include, for example, polyarteritis, rheumatoid arthritis, chronic active hepatitis, pemphigus, sarcoidosis, malignant rheumatoid arthritis, polyarteritis nodosa, and Kaplan's syndrome.
  • Malignant tumors include, for example, malignant lymphoma, Hodgkin's lymphoma, adult T-cell leukemia, mycosis fungoides, immunoblastic lymphadenitis, soft tissue sarcoma, carcinomatous lymphangiosis, paramalignant tumor syndrome, heavy chain disease , colon cancer, lung cancer, and the like.
  • bronchial asthma that is the disease to be tested is not particularly limited. All classes, grades, and stages of bronchial asthma in various classification criteria for progression of bronchial asthma can be tested. In addition, all classification categories of bronchial asthma (eg, eosinophilic asthma, atopic asthma, etc.) can be tested.
  • the subject is the target organism of the testing method of the present invention, and its species is not particularly limited.
  • Species of the subject include, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans.
  • Subjects include, for example, subjects with unknown eosinophilia, subjects already determined to have eosinophilia by another method, eosinophilia Examples include specimens that have already been determined by another method to be free of disease, specimens undergoing treatment for eosinophilia, specimens after treatment for eosinophilia, and the like.
  • the sample to be detected in step (1) is preferably extracellular vesicles of body fluid.
  • Body fluids include, for example, whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, exhaled breath, exhaled breath condensate, etc.
  • Whole blood, serum, plasma and cerebrospinal fluid are preferred, and whole blood, serum and plasma are more preferred.
  • One type of body fluid may be used alone, or two or more types may be used in combination.
  • a body fluid can be collected from a subject by a method known to those skilled in the art.
  • whole blood can be collected by blood collection using a syringe or the like.
  • Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as a supernatant after coagulation of whole blood.
  • Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when whole blood is subjected to centrifugation under non-clotting conditions.
  • blood samples blood itself such as whole blood, serum, plasma, etc., or blood-derived samples are referred to as "blood samples”.
  • Extracellular vesicles are not particularly limited as long as they are membrane vesicles that are secreted or released from cells.
  • Extracellular vesicles are usually defined as membrane vesicles that carry intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of cells, and are responsible for local and systemic intercellular communication. be.
  • Extracellular vesicles include, for example, exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles and the like.
  • Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or in accordance with known methods.
  • Methods for purifying, separating, and concentrating extracellular vesicles include, for example, ultracentrifugation (e.g., pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), methods using immunoaffinity carriers, gel filtration, field centrifugation, and the like. Flow fractionation method, FACS method and the like can be mentioned. Purification, separation, concentration and the like of extracellular vesicles can also be performed using commercially available kits. These methods may be employed singly or in combination of two or more.
  • Detection targets in step (1) include protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G ) (in the present specification, these are sometimes collectively referred to as the “protein of interest”).
  • Protein group (B) includes (B) HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2
  • Protein group (B) is a protein whose expression level in all bronchial asthma subjects is higher than that in healthy subjects.
  • protein group (B) preferably HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2
  • Protein group (C) includes (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, NucleoLysin Tia-1 IsoForm P40, NucleOlysin Tia-1 Isoform P40, NucleOlysin TIAR, DISINTEGRIN and Metalloproteinase Domain-Contening ProtEin 10, Choline T Ransport-Like Protein 2, Probable UDP-Sugar Transporter
  • Protein group (C) is a protein whose expression level in all bronchial asthma subjects is lower than that in healthy subjects.
  • the protein group (C) preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, etc., more preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2.
  • Protein group (D) includes (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxyn
  • Protein group (D) is a protein whose expression level in atopic asthmatic subjects is higher than that in healthy subjects.
  • the protein group (D) preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol Factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, etc., more preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A.
  • Protein group (E) includes (E) HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo
  • Protein group (E) is a protein whose expression level in atopic asthmatic subjects is lower than that in healthy subjects.
  • protein group (E) preferably HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, tight junction protein ZO-2, and more preferably HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase
  • Protein group (F) includes (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Tor
  • Protein group (F) is a protein whose expression level in eosinophilic asthmatic subjects is higher than that in healthy subjects.
  • the protein group (F) preferably Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, more preferably Galectin-10, Eosinophil peroxida
  • Protein group (G) includes (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptida
  • the protein group (G) is proteins whose expression level in eosinophilic asthmatic subjects is lower than that in healthy subjects.
  • the protein group (G) preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, etc., more preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4,
  • Protein group (A) is a protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil catic protein.
  • Protein group (A) is a protein whose expression level in subjects with bronchial asthma is higher than that in healthy subjects, and the number of blood eosinophils, respiratory function, tissue pathology, tissue eosinophil infiltration, clinical score , EETosis-positive eosinophil count, etc., is a particularly preferable protein as an index of severity.
  • Galectin-10 is particularly preferred.
  • Protein group (X) includes Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin-3, Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin It is a protein group consisting of heavy variable 8-61 and immunoglobulin heavy variable 9-49.
  • the protein group (X) preferably Immunoglobulin heavy variable 8-61, Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, Galectin-10, Immunoglobulin heavy variable 7-43, more preferably Immunoglobulin heavy variable 8-61 , Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, and Galectin-10, more preferably Immunoglobulin heavy variable 8-61.
  • the proteins of the protein groups (A) to (G) are, in the case of humans, proteins specified by the UniProtKB accession numbers shown in Tables 1 to 6 in Examples below. For other species, it is an orthologue of the protein identified by the UniProtKB accession numbers shown in Tables 1-6.
  • the number of target proteins in step (1) may be one, or a combination of two or more. By combining more detection targets (e.g., 2, 5, 10, 20, 30, 40, 50 or more), eosinophilia can be tested more accurately. becomes possible.
  • Detection is usually performed by measuring the amount or concentration of the target protein.
  • “Concentration” is not limited to absolute concentration, but may be relative concentration, weight per unit volume, or raw data measured to know absolute concentration.
  • the method for detecting the target protein is not particularly limited as long as it can specifically detect part or all of the target protein.
  • Specific examples of the detection method include mass spectrometry for detecting peptides constituting the target protein, and immunoassay using an antibody that specifically recognizes the target protein.
  • the amino acid sequence information of the target protein can be obtained by searching the database of EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) based on the UniProtKB accession number.
  • Immunohistochemical staining methods ELISA methods, EIA methods, RIA methods, Western blotting methods, etc. can be suitably exemplified as immunological measurement methods.
  • a peptide sample is turned into gaseous ions (ionized) using an ion source, and the peptide sample is moved in vacuum in an analysis unit and ionized using electromagnetic force or by time-of-flight difference.
  • a measurement method using a mass spectrometer that can separate and detect according to the ratio.
  • Methods of ionization using an ion source include the EI method, CI method, FD method, FAB method, MALDI method, and ESI method. and other methods can be selected as appropriate, and methods for separating the ionized peptide sample in the analysis unit include magnetic field deflection, quadrupole, ion trap, time-of-flight (TOF), and Fourier transform.
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used.
  • the sample if the sample contains phosphorylated peptides, the sample can be concentrated using iron ion immobilization affinity chromatography (Fe-IMAC) prior to sample introduction into the mass spectrometer.
  • Fe-IMAC iron ion immobilization affinity chromatography
  • peptides constituting the target protein can be separated and purified and used as a sample by liquid chromatography (LC) or HPLC.
  • the detection unit and data processing method can be selected as appropriate.
  • a peptide that has the same amino acid sequence as the peptide and is labeled with a stable isotope of known concentration is used as an internal standard. can do.
  • a stable isotope-labeled peptide one or more of the amino acids in the peptide constituting the target protein to be detected is a stable isotope-labeled peptide containing any one or more of 15N, 13C, 18O, and 2H. can be selected as appropriate, and such stable isotope-labeled peptides are produced by the F-moc method (Amblard., et al. Methods Mol Biol.
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  • the amount and/or concentration of the target protein which is a detection indicator for eosinophilia, can be provided, thereby detecting eosinophilia. It can aid in detection, assessment of severity, and the like.
  • test results obtained by the test method of the present invention including step (1) can be used as a therapeutic marker for eosinophilia therapeutic agents, elucidation of the pathology of eosinophilia, prediction of prognosis of eosinophilia, It can be used for stratification of treatment, selection of treatment method (personalized medicine, treatment response), refractory in eosinophilia, differentiation of histological type and phenotype of eosinophilia, etc. .
  • Process (2) As one aspect of the inspection method of the present invention, When the detected protein contains at least one protein (protein ABDF') selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F), moreover, (2ABDF) if the amount or concentration of the protein ABDF' detected in step (1) is equal to or higher than the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future; is preferably included. According to the testing method of the present invention including the step 2ABDF, it is possible to determine and evaluate the pathology of eosinophilia or bronchial asthma.
  • protein ABDF' protein selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F)
  • (2ABDF) if the amount
  • the detected protein contains at least one protein (protein CEG') selected from the group consisting of protein group (C), protein group (E), and protein group (G), (2CEG) if the amount or concentration of the protein CEG' detected in step (1) is less than or equal to the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future; is preferably included. According to the testing method of the present invention including step 2CEG, it is possible to determine eosinophilia.
  • the examination method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein D') selected from the protein group (D), (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • protein D' protein selected from the protein group (D)
  • (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • the examination method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein E') selected from protein group (E), (2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value; It is preferred to include
  • the testing method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein F') selected from the protein group (F), (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • protein F' protein selected from the protein group (F)
  • (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • the testing method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein G') selected from the protein group (G), (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; is preferably included.
  • protein G' protein selected from the protein group (G)
  • (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; is preferably included.
  • the testing method of the present invention when determining the presence or absence of morbidity of high-severity eosinophilia and the possibility of morbidity in the future, the testing method of the present invention, as an aspect,
  • the detected protein contains at least one protein (protein A') selected from protein group (A), (2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future; It is preferred to include
  • the cutoff value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc.
  • control subjects e.g., those suffering from eosinophilia subjects not suffering from or suffering from bronchial asthma, subjects not suffering from or suffering from atopic asthma, subjects not suffering from eosinophilic asthma
  • the amount and/or concentration of the protein of interest in extracellular vesicles or body fluids of body fluids collected from diseased subjects, subjects with low or high index values for the severity of eosinophilia, etc. can be a value determined each time or a predetermined value based on .
  • the cutoff value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles or body fluid of the body fluid collected from the reference subject or subject group (when the subject is multiple, the average value, median value), for example, 0.7 to 1.5 times. It can also be set by performing statistical analysis (more specifically, a method using Youden index is exemplified) based on Receiver Operating Characteristic (ROC) curve analysis, etc. can.
  • ROC Receiver Operating Characteristic
  • the cutoff value is, for example, the amount of the protein of interest in a past sample of the same subject. and/or concentration-based values can be used to determine therapeutic efficacy.
  • the subject was determined to be suffering from eosinophilia or bronchial asthma by the test method of the present invention including the step (2) of diagnosing eosinophilia or bronchial asthma with higher accuracy.
  • eosinophilia or bronchial asthma can be diagnosed with higher accuracy by combining the testing method of the present invention with the step of applying diagnosis of eosinophilia or bronchial asthma by a doctor. can.
  • testing method of the present invention can more accurately detect eosinophilia or bronchial asthma, combining the above steps with the testing method of the present invention can more efficiently and accurately detect "eosinophilia.” It can be diagnosed as suffering from polymorphism or bronchial asthma.
  • the test of the present invention is performed. Diagnosed as suffering from eosinophilia or bronchial asthma further to the method or as described above in "2. Diagnosis of eosinophilia or bronchial asthma with greater accuracy".
  • the test method of the present invention and the step of applying diagnosis by a doctor if On the other hand, by performing the step of treating the disease, it is possible to treat the disease in the subject.
  • the testing method of the present invention can more accurately detect eosinophilia or bronchial asthma
  • the testing method of the present invention or the step of applying the testing method of the present invention and a diagnosis by a doctor can be applied.
  • step 3 subjects suffering from eosinophilia or bronchial asthma can be treated more effectively and reliably.
  • the treatment method for eosinophilia or bronchial asthma is not particularly limited, but typically includes drug treatment.
  • Pharmaceuticals used for drug treatment are not particularly limited, but for example, ⁇ -agonists such as adrenaline and salbutamol; anticholinergics such as ipratropium and tiotropium; leukotriene modifiers such as montelukast, zafirlukast and zileuton; obesity such as cromoglycate and nedocromil cell stabilizers; corticosteroids; immunomodulators (antibody drugs) such as omalizumab, mepolizumab, reslizumab; methylxanthines such as theophylline; lidocaine; One, two, or a combination of three or more medicaments can be used.
  • Eosinophilia or bronchial asthma test agent and test kit A test agent for eosinophilia or bronchial asthma, containing at least one protein detection agent selected from the group consisting of (E), protein group (F), and protein group (G) , sometimes referred to as "the test agent of the present invention"). This will be explained below.
  • Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
  • the detection agent is not particularly limited as long as it can specifically detect the target protein.
  • the detection agent includes, for example, an antibody against the target protein.
  • the detection agent may be modified as long as its function is not significantly impaired. Modifications include, for example, addition or introduction of labels such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, colloidal gold, biotin, and the like.
  • the detection agent can also be used by immobilizing it on any solid phase.
  • the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which probes are immobilized, etc. Another example is an ELISA plate on which antibodies are immobilized, etc.).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies and the like. Examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries and other substrates. can. Immobilization of the detection agent to the solid phase is not particularly limited.
  • the antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein.
  • selectively (specifically) recognize means that the target protein can be specifically detected in, for example, Western blotting or ELISA, but is not limited thereto. Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.
  • Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or portions of the above antibodies that have antigen-binding properties, such as Fab fragments and fragments generated by Fab expression libraries.
  • the antibody of the present invention also includes an antibody that has antigen-binding to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12-11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed in Escherichia coli or the like and purified according to a standard method. Then, it is possible to immunize a non-human animal such as a rabbit and obtain the serum from the immunized animal according to a conventional method.
  • a non-human animal such as a mouse is immunized with a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein, and spleen cells obtained by immunizing a non-human animal such as a mouse are used. It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
  • the target protein used as an immunizing antigen for the production of antibodies is based on the known gene sequence information, DNA cloning, construction of each plasmid, transfection into the host, culture of the transformant, and recovery of the protein from the culture. can be obtained by the operation of These manipulations are performed according to methods known to those skilled in the art or methods described in literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). can be done.
  • a recombinant DNA capable of expressing a gene encoding a target protein in a desired host cell is prepared, introduced into the host cell for transformation, and the transformant is cultured.
  • the protein By recovering the target protein from the resulting culture, the protein can be obtained as an immunizing antigen for producing the antibody of the present invention.
  • a partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
  • the antibody of the present invention may also be prepared using an oligopeptide having a partial amino acid sequence of the target protein.
  • the oligo(poly)peptides used for the production of such antibodies need not have functional biological activity, but desirably have immunogenic properties similar to the protein of interest.
  • An oligo(poly)peptide preferably having this immunogenic property and consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
  • Antibodies against such oligo(poly)peptides can also be produced by enhancing the immunological response using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface agents such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other ingredients as needed.
  • Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain, in addition to the detection agent or the composition containing the same, those that can be used to detect the target protein in extracellular vesicles of body fluids or blood samples of a subject.
  • Specific examples of such materials include various reagents (eg, secondary antibodies, buffer solutions, etc.), instruments (eg, extracellular vesicle purification, separation, and concentration instruments (eg, columns, etc.)), and the like.
  • Prophylactic or therapeutic agent for eosinophilia or bronchial asthma a group consisting of protein group (A), protein group (B), protein group (D), and protein group (F) and at least one protein enhancer selected from the group consisting of protein group (C), protein group (E), and protein group (G). It relates to a prophylactic or therapeutic agent for eosinophilia or bronchial asthma (in this specification, sometimes referred to as "the drug of the present invention") containing at least one drug selected from the above. This will be explained below.
  • Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
  • Inhibitors include, for example, antibodies against target proteins.
  • the antibody the same antibody as explained in the above “4. Test agent and test kit for eosinophilia or bronchial asthma” can be used.
  • Another example of an inhibitor is a target protein expression inhibitor.
  • the agent for suppressing the expression of the target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, its precursor, etc.
  • examples include gene-specific small interfering RNA (siRNA) of the target protein, target Gene-specific microRNA (miRNA) for proteins, gene-specific antisense nucleic acids for target proteins, expression vectors for these; gene-specific ribozymes for target proteins; gene editing agents for target proteins by CRISPR/Cas system, etc.
  • the expression suppression means that the expression level of the target protein, its mRNA, etc. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and also includes setting the expression level to zero.
  • Enhancers include, for example, target protein expression enhancers.
  • the agent for enhancing the expression of the target protein is not particularly limited as long as it can enhance the expression level of the target protein, its mRNA, its precursor, etc.
  • Examples include expression vectors for the target protein.
  • expression enhancement refers to enhancing the expression level of the target protein, its mRNA, etc., for example, 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, 1000, 10000 times. means.
  • the gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein.
  • the siRNA is preferably, for example, 18 bases or longer, 19 bases or longer, 20 bases or longer, or 21 bases or longer.
  • the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. Any combination of the upper and lower limits of the siRNA length described herein is assumed.
  • siRNA may be shRNA (small hairpin RNA).
  • a portion of shRNA can be designed to form a stem-loop structure.
  • sequence a if the sequence of a certain region is sequence a, and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand in the order of sequence a, spacer, and sequence b. and can be designed to be 45-60 bases in length overall.
  • Sequence a is a sequence of a partial region of the base sequence encoding the target protein of interest, and the target region is not particularly limited, and any region can be a candidate.
  • the length of sequence a is 19-25 bases, preferably 19-21 bases.
  • the gene-specific siRNA of the target protein may have additional bases at the 5' or 3' end.
  • the length of the additional bases is usually about 2-4 bases.
  • the additional base may be either DNA or RNA, but using DNA may improve the stability of the nucleic acid.
  • Such additional base sequences include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', tttt-3 ', uuuuuu-3', and the like, but are not limited to these.
  • the siRNA may have an overhang sequence (overhang) at the 3' end, and specifically includes those with dTdT (dT represents deoxythymidine) added. It may also be a blunt end (blunt end) without terminal addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand.
  • aiRNA asymmetrical interfering RNA
  • a typical aiRNA consists of an antisense strand of 21 nucleotides, a sense strand of 15 nucleotides, and an overhang structure of 3 nucleotides at each end of the antisense strand.
  • the position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, the target sequence is from the 5'-UTR and the start codon to about 50 bases and from a region other than the 3'-UTR. Sequence selection is preferred. BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) to confirm the specificity of the selected target sequence.
  • a sense strand having a TT or UU 3' overhang at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a UU 3'-terminal overhang may be designed as an siRNA.
  • siRNA which is a precursor of siRNA
  • an arbitrary linker sequence (for example, about 5 to 25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and antisense strand are connected via the linker sequence. It can be designed by concatenating.
  • siRNA and/or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include the following. siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp/techlib/misc/psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
  • siRNA is prepared by synthesizing the sense strand and antisense strand of the target sequence on mRNA with an automatic DNA/RNA synthesizer, and denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute. It can be prepared by annealing at about 30 to about 70° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with the RNA-cleaving protein dicer.
  • the gene-specific miRNA for the target protein is optional as long as it inhibits translation of the gene encoding the target protein.
  • miRNAs may bind to the 3' untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA as siRNAs do.
  • miRNA may be pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), or mature miRNA.
  • the length of miRNA is not particularly limited, and the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50-80 bases, and the length of mature miRNA is usually 18 bases. ⁇ 30 bases.
  • the gene-specific miRNA of the protein of interest is preferably pre-miRNA or mature miRNA, more preferably mature miRNA.
  • Such gene-specific miRNAs of target proteins may be synthesized by known methods or purchased from companies that provide synthetic RNAs.
  • a gene-specific antisense nucleic acid for a target protein is a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the gene encoding the target protein, or a part thereof, and is specific to the mRNA. It is a nucleic acid that has the function of inhibiting target protein synthesis by forming and binding to a stable double strand.
  • Antisense nucleic acids can be DNA, RNA, or DNA/RNA chimeras. When the antisense nucleic acid is DNA, an RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) to cause selective degradation of the target RNA.
  • RNase H endogenous ribonuclease H
  • the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of interest protein.
  • the intron sequence can be determined by comparing the genomic sequence and the cDNA nucleotide sequence of the gene of the target protein using homology search programs such as BLAST and FASTA.
  • the target region of the gene-specific antisense nucleic acid of the target protein is not limited in length as long as the hybridization of the antisense nucleic acid results in inhibition of translation into the target protein.
  • a gene-specific antisense nucleic acid for a protein of interest may be the entire sequence or a partial sequence of the mRNA encoding the protein of interest. Oligonucleotides of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred in consideration of ease of synthesis, antigenicity, intracellular translocation, etc., but are not limited to these.
  • the 3' end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA or initial transcript of the target protein gene to inhibit translation into protein, but also binds to these genes, which are double-stranded DNA. It may be one that can form a triplex and inhibit transcription to RNA (antigene).
  • nucleotide molecules that make up the gene-specific siRNA of the protein of interest, the gene-specific miRNA of the protein of interest, and the gene-specific antisense nucleic acid of the protein of interest described above have stability (chemical and/or enzymatic) and specific activity ( Various chemical modifications may be included in order to improve affinity with RNA).
  • the phosphate residue (phosphate) of each nucleotide that constitutes the antisense nucleic acid is replaced with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithioate. can be substituted with chemically modified phosphate residues such as phosphorodithioates.
  • the base moiety pyrimidine, purine
  • part of the nucleotide molecules that constitute siRNA or miRNA may be replaced with natural DNA.
  • Gene-specific siRNAs for proteins of interest, gene-specific miRNAs for proteins of interest, and gene-specific antisense nucleic acids for proteins of interest, etc. target mRNA or early transcripts based on the cDNA or genomic DNA sequence of the gene for the protein of interest. It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA/RNA automatic synthesizer. In addition, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known techniques.
  • the expression vector comprises a promoter sequence and a gene-specific siRNA for the protein of interest, a gene-specific miRNA for the protein of interest, a gene-specific antisense nucleic acid for the protein of interest, or a coding sequence for the protein of interest (optionally and a transcription termination signal sequence), optionally other sequences.
  • the promoter is not particularly limited, for example, RNA polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter; mouse and human U6-snRNA promoters; Examples thereof include RNA polymerase III (polIII) promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter.
  • Other sequences are not particularly limited, and various known sequences that can be contained in an expression vector can be employed. Examples of such sequences include origins of replication, drug resistance genes, and the like.
  • the types of drug-resistant genes and the types of vectors can be exemplified by those described above.
  • ribozyme in the narrow sense means RNA having enzymatic activity to cleave nucleic acid, but in the present application, it also includes DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acids are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzymatic activity at about 40 bases, and a few bases at each end (about 10 bases in total) adjacent to the part that forms the hammerhead structure are linked to the sequence complementary to the desired cleavage site of the mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is converted to a single strand by using a hybrid ribozyme that ligates an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • a hybrid ribozyme that ligates an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • the application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. .
  • the form of the drug of the present invention is not particularly limited, and can take any form commonly used for each application depending on the use of the drug of the present invention.
  • Forms include, for example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops, etc.) when the application is pharmaceuticals, health-promoting agents, nutritional supplements (supplements, etc.), etc. ), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly preparations suitable for oral intake forms (oral formulations), formulations suitable for parenteral intake such as nasal drops, inhalants, rectal suppositories, inserts, enemas, jelly, injections, patches, lotions, cream oral dosage forms).
  • liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants , cake mixes, powdered or liquid dairy products, breads, cookies, etc.
  • liquid for example, liquid (solution, milky lotion, suspension, etc.), semisolid (gel, cream, paste, etc.), solid (tablet, granule, capsule, Film agent, kneaded product, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid dentifrice, liquid dentifrice, toothpaste, etc.), mouthwash, Coating agents, patches, mouth fresheners, foods (eg, chewing gum, tablet candy, candy, gummies, films, lozenges, etc.) and the like.
  • the drug of the present invention may further contain other ingredients as necessary.
  • Other ingredients are not particularly limited as long as they are ingredients that can be blended in pharmaceuticals, food compositions, oral compositions, health-promoting agents, nutritional supplements (supplements, etc.). , carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, chelating agents and the like.
  • the total content of the suppressor and enhancer for the target protein of the drug of the present invention depends on the type of suppressor and enhancer, application, mode of use, application target, condition of application target, etc., and the limitation is However, it can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.
  • the amount of application (for example, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits efficacy. mg/kg body weight.
  • the above dosage is preferably administered once or divided into 2 to 3 times a day, and can be adjusted appropriately according to age, condition and symptoms.
  • a screening method for an active ingredient of a preventive or therapeutic agent for eosinophilia or bronchial asthma Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), and Protein Group (G)
  • a screening method for an active ingredient (or a candidate substance thereof) of a prophylactic or therapeutic agent for eosinophilia or bronchial asthma using the amount or concentration of at least one protein as an indicator (herein referred to as "the (also referred to as “active ingredient screening method”). This will be explained below.
  • Hyperacidophilia or bronchial asthma measurement of the amount or concentration of the target protein, etc. are defined in the above "1. Test method for eosinophilia or bronchial asthma”.
  • animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
  • test substances can be used, regardless of whether they are naturally occurring compounds or artificially created compounds.
  • purified compounds but also compositions in which various compounds are mixed, and extracts of animals and plants can be used.
  • Compounds include not only low-molecular-weight compounds, but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.
  • the active ingredient screening method of the present invention comprises at least one selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F).
  • a test substance is eosinophilic if the value of the indicator for the protein is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance.
  • the test substance is eosinophilic if the value of the indicator for the protein is higher than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance. It comprises at least one step selected from the group consisting of the steps of selecting an active ingredient for a prophylactic or therapeutic agent for bloating or bronchial asthma.
  • a corresponding protein means the same protein as the target protein used as an index.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • High means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
  • a method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma is characterized by: At least selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G)
  • a method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma using the amount or concentration of one kind of protein as an index in the present specification, it may be referred to as the "toxicity evaluation method of the present invention" .
  • the toxicity evaluation method of the present invention includes at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F). is greater than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance Determining proliferative disease or bronchial asthma provocation or exacerbation, and at least one protein selected from the group consisting of protein group (C), protein group (E), and protein group (G) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance It comprises at least one step selected from the group consisting of the steps of judging proliferative or exacerbating polycythemia or bronchial asthma.
  • a corresponding protein means the same protein as the target protein used as an index.
  • High means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • Test example 1 Preparation of extracellular vesicle fraction Serum from 8 human subjects diagnosed with bronchial asthma (4 with eosinophilic asthma, 4 with atopic asthma) and 4 healthy human subjects ) Extracellular vesicle fractions were prepared from each serum. The extracellular vesicle fraction was prepared using an extracellular vesicle preparation kit (MagCapturTM Exosome Isolation Kit PS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with the same volume of serum for each sample. Ta.
  • MagCapturTM Exosome Isolation Kit PS manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • the extracellular vesicle particle count and particle size were measured for the obtained extracellular vesicle fraction. Specifically, it was measured using Nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size. The particle size (hydrodynamic diameter) in can be calculated.
  • extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was carried out as follows. 5 to 8 ⁇ L of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to allow the extracellular vesicles to settle on the formvar of the grid. After washing with PBS three times, a blocking reaction (1% BSA/PBS, 10 minutes) was performed, followed by a primary antibody reaction (Invitrogen AHS0902 Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2/57, 100-fold dilution, 2.5 hours, room temperature).
  • a blocking reaction 1% BSA/PBS, 10 minutes
  • extracellular vesicle fractions were Western blotted using antibodies against exosome markers (anti-Flotillin-1 antibody, anti-CD9 antibody, and anti-CD81 antibody).
  • Test example 2 Proteomics analysis (DIA proteome analysis) Proteins in the extracellular vesicle fraction obtained in Test Example 1 were quantified by DIA proteome analysis. Specifically, it was carried out as follows.
  • Digested peptides were precipitated, acidified with 30 ⁇ L of 5% trifluoroacetic acid to remove detergent, and samples were centrifuged at 17,400 ⁇ g for 15 minutes. The supernatant was desalted using a C18-StageTip, dried using a centrifugal evaporator, and the dried peptide was dissolved in 3% ACN and 0.1% FA.
  • Peptides were injected directly into a 75 ⁇ m ⁇ 20 cm PicoFrit emitter (New Objective) packed with C18 core-shell particles (CAPCELL CORE MP 2.7 ⁇ m, 160 ⁇ material; Osaka Soda Co., Ltd.) at 50 °C and analyzed using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) at a flow rate of 100 nl/min with a gradient of 90 minutes.
  • MS1 spectra were collected at a resolution of 30,000 over the range 495-785 m/z to set an automatic gain control target of 3e6 and a maximum injection time of 55.
  • MS2 collected spectra over 200 m/z at a resolution of 15,000 to set the automatic gain control target to 3e6, the maximum injection time to "auto", and the stepped normalized collision energies to 24, 26, and 28%.
  • the MS2 isolation width was set to 4 m/z and the 500-780 m/z overlapping window pattern used the optimized window placement in Skyline (PMID: 20147306).
  • MS files were searched against the mouse spectral library using Scaffold DIA (Proteome Software, Inc., Portland, OR).
  • Mouse spectral libraries were generated from the human protein sequence database (UniProt id UP000005640, reviewed, canonical) by Prosit (PMID: 32214105, PMID: 31133760).
  • the search parameters for Scaffold DIA were: experimental data search enzyme, trypsin, maximum deletion cleavage site, 1, precursor mass tolerance, 8 ppm, fragment mass tolerance, 10 ppm, static modification, cysteine carbamidomethylation.
  • the protein identification threshold was set to less than 1% for both peptide and protein false discovery rates.
  • Peptide quantification was calculated with Scaffold DIA's EncyclopeDIA algorithm (PMID: 30510204). For each peptide, the four highest quality fragment ions were selected and quantified. Protein quantification was estimated from summed peptide quantification.
  • Proteins serving as biomarkers for eosinophilia or bronchial asthma were extracted from this according to the following criteria.
  • Table 2 shows proteins whose expression levels in all bronchial asthma subjects were lower than in healthy subjects.
  • "Fold change” is the value obtained by dividing the average expression level of healthy subjects by the average expression level of all subjects with bronchial asthma (average expression level of healthy subjects / expression level of all subjects with bronchial asthma). amount average value).
  • Table 5 shows proteins whose expression levels in subjects with eosinophilic asthma were higher than those in healthy subjects, among the extracted biomarkers for eosinophilia or bronchial asthma.
  • "Fold change” is the value obtained by dividing the average expression level of subjects with eosinophilic asthma by the average expression level of healthy subjects (mean expression level of subjects with eosinophilic asthma/ The expression level average value of the subject) is shown.
  • Galectin-10 Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, and Non-secretory ribonuclease could be used to differentiate from atopic asthma (Fig. 4). .
  • Test example 3 Evaluation of eosinophilia or bronchial asthma biomarkers1 Galectin-10, bone marrow proteoglycan, eosinophil peroxidase, non-secretory ribonuclease, and eosinophil cationic protein expression levels of eosinophilia or bronchial asthma biomarkers and blood eosinophils in each sample of Test Example 1 Based on the numbers, correlation coefficients between the expression levels of eosinophilia or bronchial asthma biomarkers and the number of eosinophils were calculated.
  • the correlation coefficient between the expression level of Galectin-10 and the eosinophil count was 0.95
  • the correlation coefficient between the expression level of bone marrow proteoglycan and the eosinophil count was 0.93
  • the expression level of eosinophil peroxidase was 0.93
  • the eosinophil count was 0.88
  • the correlation coefficient between the expression level of non-secretory ribonuclease and the eosinophil count was 0.88
  • the correlation coefficient between the expression level of eosinophil catic protein and the eosinophil count was 0.88.
  • the correlation coefficient was 0.44.
  • Test example 4 Evaluation of eosinophilia or bronchial asthma biomarkers2 Based on the quantitative results of Test Example 2, an ROC curve was prepared when Galectin-10 was used alone as a biomarker for bronchial asthma. Also, as a positive control, an ROC curve was prepared for the same subject using the blood eosinophil count as a bronchial asthma biomarker. Specifically, an ROC curve with sensitivity (positive rate) on the vertical axis and a value obtained by subtracting specificity from 1 (1 - specificity) (false positive rate) on the horizontal axis was created using the statistical software JMP. .
  • the AUC was 0.96875 when Galectin-10 was used as a biomarker, and the AUC was 0.93750 when the blood eosinophil count was used as a biomarker. From this, it was found that galectin-10 has a higher diagnostic ability for asthma than the eosinophil count.
  • Test example 5 Evaluation of eosinophilia or bronchial asthma biomarkers3
  • the correlation coefficient between the expression level of biomarkers for eosinophilia or bronchial asthma such as Galectin-10, blood eosinophil count, and respiratory function (FEV1%) was calculated for each sample in Test Example 1. .
  • the correlation coefficient between the expression level of Galectin-10 and FEV1% was -0.8344 (p-value 0.0014), and the correlation coefficient between eosinophil count and FEV1% was -0.8073 (p-value 0.0027).
  • Ta Other eosinophilia or bronchial asthma biomarkers were also correlated with FEV1%.
  • Test example 6 Evaluation of eosinophilia or bronchial asthma biomarkers4 Refractory asthma may be complicated by eosinophilic sinusitis. Eosinophilic sinusitis is classified into chronic rhinosinusitis with nasal polyp (CRSwNP), which has a poor prognosis, and chronic rhinosinusitis without nasal polyp (CRSwNP), which has a favorable prognosis. polyp: CRSsNP). Therefore, we investigated whether it is possible to distinguish between these by eosinophilia or bronchial asthma biomarkers.
  • Extracellular vesicle fractions (Exosomes) and tissues (inferior turbinate and polyp) were collected from 7 CRSwNP human subjects and 6 CRSsNP human subjects in the same manner as in Test Example 1, and Galectin- Expression levels of eosinophilia or bronchial asthma biomarkers such as 10 were measured. The results are shown in FIGS. 1 and 2.
  • FIG. Galectin-10 was found to be increased in exosome and tissue therapy of CRSwNP.
  • the correlation coefficient between the number of blood eosinophils and the Galectin-10 expression level in tissues, and the correlation coefficient between the Galectin-10 expression level in exosomes and the Galectin-10 expression level in tissues were calculated.
  • the correlation coefficient between blood eosinophil count and Galectin-10 expression level in tissues was 0.5409 (p-value 0.0858), indicating that Galectin-10 expression level in exosomes and Galectin-10 expression level in tissues was 0.6709 (p-value 0.0337). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with the Galectin-10 expression level in tissues rather than the blood eosinophil count.
  • the correlation coefficient between blood eosinophil count and tissue eosinophil infiltration (/HPF), and the correlation coefficient between Galectin-10 expression level in exosomes and tissue eosinophil infiltration (/HPF) Calculated.
  • the correlation coefficient between the number of blood eosinophils and tissue eosinophil infiltration (/HPF) was 0.6053 (p-value 0.0284)
  • the expression level of Galectin-10 in exosomes and tissue eosinophil infiltration (/ HPF) was 0.7534 (p-value 0.0119). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with tissue eosinophil infiltration rather than the blood eosinophil count.
  • Test example 7 Evaluation of eosinophilia or bronchial asthma biomarkers5 It has been reported that suppression of EETosis suppresses exacerbation of asthma in a mouse asthma model (Nature Cell Biology volume 23, pages 1060-1072 (2021)). Therefore, for the subject of Test Example 6, the number of EETosis-positive eosinophils (/HPF) in the tissue was measured according to the method described in the above document, and the number of blood eosinophils and the expression level of Galectin-10 in exosomes The correlation coefficient of was calculated.
  • the correlation coefficient between the number of blood eosinophils and the number of EETosis-positive eosinophils in tissues was 0.5522 (p value 0.0504).
  • the correlation coefficient with EETosis-positive eosinophil count (/HPF) was 0.7886 (p-value 0.0067). From this, it was found that the Galectin-10 expression level in exosomes correlated more strongly with the number of EETosis-positive eosinophils than the number of blood eosinophils.
  • Test example 8 Evaluation of eosinophilia or bronchial asthma biomarkers6
  • eosinophil activation increases Galectin-10 in exosomes Specifically, according to previous reports (Nat Protoc. 2006; 1(6): 2613-20., Blood. 2013 Mar 14; 121(11): 2074-83.), eosinophils were isolated and EETosis was induced. After that, Western blotting of supernatant-derived exosomes was performed.
  • Galectin-10 was also detectable by immunoblotting, and it was found that asthma, normal, and COPD could be determined.
  • Galectin-10 was found to increase in asthmatic lung tissue.

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Abstract

The present invention addresses the problem of providing a biomarker of eosinophilia or bronchial asthma, and a method for using the biomarker. Said problem is solved by a method for testing eosinophilia or bronchial asthma, the method comprising: (1) a step for detecting at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F) and protein group (G) in extracellular vesicles of a body fluid or in the body fluid collected from a subject.

Description

好酸球増多症又は気管支喘息バイオマーカーEosinophilia or bronchial asthma biomarkers
　本発明は、好酸球増多症又は気管支喘息バイオマーカー等に関する。 The present invention relates to eosinophilia or bronchial asthma biomarkers and the like.
　気管支喘息は、「気道の慢性炎症を本態とし、変動性をもった気道狭窄や咳などの臨床症状で特徴づけられる疾患」と定義される。近年の調査では、日本において継続的に医療を受けている喘息の総患者数は100万人以上とされており、また全世界では喘息の総患者数は約3億人とされている。喘息には明確な診断基準が存在せず、喘息予防・管理ガイドラインでは喘息診断の目安が定められている。発作性の呼吸困難や咳が反復することに加え、可逆性の気流制限、気道過敏性の亢進、アトピー素因の存在、気道炎症の存在の有無を参考にすることが推奨されており、これらの指標を総合的に判断して診断する必要がある。現在喘息の診断に用いられているバイオマーカーとして、血中及び喀痰好酸球数、呼気一酸化窒素濃度（FeNO）、血清IgE値が挙げられるが、これらのみで喘息の有無を判断することは困難である。 Bronchial asthma is defined as "a disease characterized by clinical symptoms such as fluctuating airway narrowing and coughing, characterized by chronic inflammation of the airways." According to a recent survey, the total number of asthma patients receiving continuous medical care in Japan is more than 1 million, and the total number of asthma patients in the world is about 300 million. There are no clear diagnostic criteria for asthma, and guidelines for asthma diagnosis are defined in the Asthma Prevention and Management Guidelines. In addition to repeated paroxysmal dyspnea and coughing, it is recommended to refer to the presence of reversible airflow limitation, increased airway hyperresponsiveness, presence of atopic predisposition, and presence of airway inflammation. It is necessary to make a diagnosis by comprehensively judging the indicators. Biomarkers currently used to diagnose asthma include blood and sputum eosinophil counts, exhaled nitric oxide (FeNO), and serum IgE levels. Have difficulty.
　また、近年では難治性喘息に対する抗体治療が開発されており、注目を集めている。高用量の吸入ステロイドと長時間作用型β2刺激薬（LABA）のみではコントロール不良であり、加えて他の治療が必要である喘息を難治性喘息と呼ぶ。このような患者では抗IgE抗体、抗IL-5抗体、抗Il－5受容体α抗体、抗IL-4状態α抗体などが使用可能となっているが、これらの使い分けを判断したり、効果を予測したりするためのバイオマーカーは上記の3つ以外には存在しない。そのため、治療選択、効果判定などに有用な新規バイオマーカーの開発が求められている。 In addition, in recent years, an antibody treatment for intractable asthma has been developed and is attracting attention. Asthma that is poorly controlled with high-dose inhaled corticosteroids and long-acting β2-agonists (LABAs) alone and that requires other treatments is called refractory asthma. Anti-IgE antibody, anti-IL-5 antibody, anti-IL-5 receptor α antibody, anti-IL-4 status α antibody, etc. can be used in such patients. There are no other biomarkers other than the three mentioned above for predicting Therefore, the development of novel biomarkers useful for treatment selection, effect determination, etc. is required.
　現在、喘息のバイオマーカーとして、各オミクスから様々な候補が挙げられているが、単一のマーカーとして有効性が広く確認されているものはない。バイオマーカーとして特に注目されているのは、表現型に最も近いオミクスであるプロテオミクスである。しかし、タンパク質は核酸と違い増幅ができず、網羅的な解析系が確立していないといった点から、疾患を対象としたプロテオミクス研究は遅れているのが現状である。 Currently, various omics have proposed various candidates as biomarkers for asthma, but none have been widely confirmed to be effective as a single marker. Proteomics, which is the omics closest to the phenotype, is of particular interest as a biomarker. However, unlike nucleic acids, proteins cannot be amplified, and a comprehensive analysis system has not been established. Therefore, proteomics research for diseases is currently lagging behind.
　血清は、非侵襲的に繰り返し採取可能であるので理想的な検査試料と考えられるものの、そこに含まれる99%以上のタンパク質はバイオマーカーの観点から夾雑物とされる多量タンパク（アルブミン等）であるため、プロテオミクスの観点からは、検査資料としての有用性は低い。例えば、COPDと喘息を鑑別可能なバイオマーカーに関する最近の論文（非特許文献１）では、患者数十人の解析を試みるも、解析が2次元電気泳動法による古い質量分析(MS)技術によるものであっただけでなく、大量夾雑物を含む血清を用いたものであったため、見出されたバイオマーカーは Serum is considered an ideal test sample because it can be collected non-invasively and repeatedly. Therefore, from the viewpoint of proteomics, its usefulness as test data is low. For example, a recent paper on biomarkers that can distinguish between COPD and asthma (Non-Patent Document 1) attempted to analyze dozens of patients, but the analysis was based on an old mass spectrometry (MS) technique based on two-dimensional electrophoresis. The biomarkers found were not only
　本発明は、好酸球増多症又は気管支喘息のバイオマーカー及びその利用方法を提供することを課題とする。 An object of the present invention is to provide a biomarker for eosinophilia or bronchial asthma and a method for using the same.
　本発明者は、鋭意研究を進めた結果、被検体から採取された体液の細胞外小胞又は体液における特定のタンパク質群が、好酸球増多症又は気管支喘息のバイオマーカーとして有用であることを見出した。この知見に基づいてさらに研究を進めた結果、本発明が完成した。即ち、本発明は、下記の態様を包含する。 As a result of intensive research, the present inventors found that extracellular vesicles in body fluids collected from subjects or specific protein groups in body fluids are useful as biomarkers for eosinophilia or bronchial asthma. I found As a result of further research based on this knowledge, the present invention was completed. That is, the present invention includes the following aspects.
　項１．　好酸球増多症又は気管支喘息を検査する方法であって、
（1）被検体から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
（A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質を検出する工程を含む、検査方法。 Section 1. A method of testing for eosinophilia or bronchial asthma, comprising:
(1) Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D), protein in extracellular vesicles of body fluid collected from a subject or body fluid Group (E), Protein Group (F), and Protein Group (G):
(A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
(B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
(C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
(D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
(E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
(F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
A testing method comprising the step of detecting at least one protein selected from the group consisting of
　項２．　検査対象疾患が呼吸器疾患である、項１に記載の検査方法。 Section 2. Item 1, wherein the disease to be tested is a respiratory disease.
　項３．　検査対象疾患が気管支喘息である、項１又は２に記載の検査方法。 Section 3. Item 1 or 2, wherein the disease to be tested is bronchial asthma.
　項４．　検査対象疾患が好酸球性喘息である、項１～３のいずれかに記載の検査方法。 Section 4. The examination method according to any one of Items 1 to 3, wherein the disease to be examined is eosinophilic asthma.
　項５．　検出するタンパク質が、タンパク質群（F）及びタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質を含む、項４に記載の検査方法。 Item 5. Item 4, wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (F) and protein group (G).
　項６．　検出したタンパク質が、タンパク質群（F）より選択される少なくとも1種のタンパク質（タンパク質F’）を含む場合、さらに、
（2F）前記工程（1）で検出されたタンパク質F’の量又は濃度がカットオフ値以上である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
を含む、項４又は５に記載の検査方法。 Item 6. When the detected protein contains at least one protein (protein F') selected from the protein group (F),
(2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to Item 4 or 5, comprising:
　項７．　検出したタンパク質が、タンパク質群（G）より選択される少なくとも1種のタンパク質（タンパク質G’）を含む場合、さらに、
（2G）前記工程（1）で検出されたタンパク質G’の量又は濃度がカットオフ値以下である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
を含む、項４～６のいずれかに記載の検査方法。 Item 7. When the detected protein contains at least one protein (protein G') selected from the protein group (G),
(2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value;
7. The inspection method according to any one of items 4 to 6.
　項８．　検査対象疾患がアトピー性喘息である、項１～３のいずれかに記載の検査方法。 Item 8. The examination method according to any one of items 1 to 3, wherein the disease to be examined is atopic asthma.
　項９．　検出するタンパク質が、タンパク質群（D）及びタンパク質群（E）からなる群より選択される少なくとも1種のタンパク質を含む、項８に記載の検査方法。 Item 9. Item 8, wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (D) and protein group (E).
　項１０．　検出したタンパク質が、タンパク質群（D）より選択される少なくとも1種のタンパク質（タンパク質D’）を含む場合、さらに、
（2D）前記工程（1）で検出されたタンパク質D’の量又は濃度がカットオフ値以上である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
を含む、項８又は９に記載の検査方法。 Item 10. When the detected protein contains at least one protein (protein D') selected from the protein group (D),
(2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to Item 8 or 9, comprising:
　項１１．　検出したタンパク質が、タンパク質群（E）より選択される少なくとも1種のタンパク質（タンパク質E’）を含む場合、さらに、
（2E）前記工程（1）で検出されたタンパク質E’の量又は濃度がカットオフ値以下である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
を含む、項８～１０のいずれかに記載の検査方法。 Item 11. When the detected protein contains at least one protein (protein E') selected from protein group (E),
(2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value;
The inspection method according to any one of Items 8 to 10, comprising
　項１２．　検出するタンパク質が、タンパク質群（A）を含む、項１～１１のいずれかに記載の検査方法。 Item 12. The inspection method according to any one of items 1 to 11, wherein the proteins to be detected include protein group (A).
　項１３．　検出したタンパク質が、タンパク質群（A）より選択される少なくとも1種のタンパク質（タンパク質A’）を含む場合、さらに、
（2A）前記工程（1）で検出されたタンパク質A’の量又は濃度がカットオフ値以上である場合に、前記被検体が高重症度の好酸球増多症又は気管支喘息に罹患している又は高重症度の好酸球増多症又は気管支喘息に将来罹患する可能性が高いと判定する工程、
を含む、項１～１２のいずれかに記載の検査方法。 Item 13. When the detected protein contains at least one protein (protein A') selected from protein group (A),
(2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future;
The inspection method according to any one of Items 1 to 12, comprising:
　項１４．　タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
（A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、好酸球増多症又は気管支喘息の検査薬。 Item 14. Protein Group (A), Protein Group (X), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), and Protein Group (G):
(A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
(B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
(C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
(D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
(E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
(F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
A test agent for eosinophilia or bronchial asthma, comprising at least one protein-detecting agent selected from the group consisting of:
　項１５．　タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（D）、並びにタンパク質群（F）：
（A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、並びに
（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質の抑制剤、並びに
タンパク質群（C）、タンパク質群（E）、並びにタンパク質群（G）：
（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、並びに
（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質の亢進剤
からなる群より選択される少なくとも1種の薬剤を含有する、好酸球増多症又は気管支喘息の予防又は治療剤。 Item 15. Protein Group (A), Protein Group (X), Protein Group (B), Protein Group (D), and Protein Group (F):
(A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
(B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
(D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- Protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2, and (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease , Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig -like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum-Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A protein group,
An inhibitor of at least one protein selected from the group consisting of, as well as protein group (C), protein group (E), and protein group (G):
(C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
(E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, Protein group consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, Synaptotagmin-like protein 4, and charged multivesicular body protein 2a, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ- associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein , STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 A protein group consisting of member 4, Solute carrier family 12 member 7, and Solute carrier family 43 member 3,
A prophylactic or therapeutic agent for eosinophilia or bronchial asthma, containing at least one drug selected from the group consisting of at least one protein enhancer selected from the group consisting of:
　項１６．　被検物質で処理された動物から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
（A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の予防又は治療剤の有効成分のスクリーニング方法。 Item 16. Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D ), Protein Group (E), Protein Group (F), and Protein Group (G):
(A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
(B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
(C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
(D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
(E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
(F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
A method of screening for an active ingredient of a prophylactic or therapeutic agent for eosinophilia or bronchial asthma, wherein the amount or concentration of at least one protein selected from the group consisting of is used as an indicator.
　項１７．　被検物質で処理された動物から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
（A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の誘発性又は増悪性の評価方法。 Item 17. Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D ), Protein Group (E), Protein Group (F), and Protein Group (G):
(A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
(B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
(C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
(D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
(E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
(F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
A method for evaluating provocation or exacerbation of eosinophilia or bronchial asthma, wherein the amount or concentration of at least one protein selected from the group consisting of is used as an index.
　本発明によれば、好酸球増多症又は気管支喘息のバイオマーカーを提供することができる。該バイオマーカーを利用することにより、好酸球増多症又は気管支喘息の検査、好酸球増多症又は気管支喘息の予防又は治療、好酸球増多症又は気管支喘息の予防又は治療剤の有効成分のスクリーニング、好酸球増多症又は気管支喘息の誘発性又は増悪性の評価等が可能になり得る。 According to the present invention, biomarkers for eosinophilia or bronchial asthma can be provided. By using the biomarker, it is possible to test for eosinophilia or bronchial asthma, prevent or treat eosinophilia or bronchial asthma, and use a preventive or therapeutic agent for eosinophilia or bronchial asthma. Screening of active ingredients, evaluation of inducing or aggravating eosinophilia or bronchial asthma, and the like may become possible.
試験例6の細胞外小胞画分（Exosome）におけるGalectin-10発現量の測定結果を示す。1 shows the measurement results of Galectin-10 expression levels in extracellular vesicle fractions (Exosomes) in Test Example 6. FIG. 試験例6の組織（下鼻甲介及びpolyp）におけるGalectin-10発現量の測定結果を示す。1 shows the measurement results of Galectin-10 expression level in tissues (inferior nasal turbinate and polyp) of Test Example 6. FIG. 試験例8の、好酸球活性化処理の有無（IL-5+PAF (+)/(-)）における、エクソソーム中及び細胞ライセート中のGalectin-10量の測定結果を示す。Fig. 8 shows measurement results of Galectin-10 amounts in exosomes and cell lysates with and without eosinophil activation treatment (IL-5+PAF (+)/(-)) in Test Example 8. 試験例2のプロテオミクス解析の測定結果を示す。各グラフ情報に測定対象のタンパク質を示す。縦軸は血清エクソソーム中の発現レベルを示す。横軸中、HCは健常被検体を示し、ABAはアトピー性喘息被検体を示し、EBAは好酸球性喘息被検体を示す。2 shows the measurement results of proteomics analysis in Test Example 2. FIG. Each graph information shows the protein to be measured. The vertical axis indicates the expression level in serum exosomes. On the horizontal axis, HC indicates healthy subjects, ABA indicates atopic asthmatic subjects, and EBA indicates eosinophilic asthmatic subjects. 試験例9のイムノブロッティングの結果を示す。HCは健常被検体を示し、COPDはCOPD被検体を示し、BAは喘息被検体を示す。2 shows the results of immunoblotting in Test Example 9. FIG. HC indicates healthy subjects, COPD indicates COPD subjects, and BA indicates asthmatic subjects. 試験例10の蛍光免疫染色の結果を示す。HCは健常被検体を示し、BAは喘息被検体を示す。2 shows the results of fluorescent immunostaining in Test Example 10. FIG. HC indicates healthy subjects and BA indicates asthmatic subjects. 試験例11のプロテオミクスの結果を示す。HCは健常被検体を示し、BAは喘息被検体を示す。2 shows the proteomics results of Test Example 11. FIG. HC indicates healthy subjects and BA indicates asthmatic subjects. 気管支壁肥厚の程度（WA％）の測定・算出方法を示す。A method for measuring and calculating the degree of bronchial wall thickening (WA%) is shown. 試験例12の血清エクソソーム中のGalectin-10の発現レベル及び血中好酸球数と気管支壁肥厚の程度（WA％）との相関を示す。12 shows the correlation between the expression level of Galectin-10 in serum exosomes in Test Example 12, the blood eosinophil count, and the degree of bronchial wall thickening (WA%). 試験例12の血清エクソソーム中のGalectin-10の発現レベル及び血中好酸球数と呼吸機能（FEV1％）との相関を示す。The expression level of Galectin-10 in serum exosomes of Test Example 12 and the correlation between blood eosinophil count and respiratory function (FEV1%) are shown. 試験例6の、細胞外小胞画分と組織間での発現量の相関係数（横軸）を示す。FIG. 10 shows the correlation coefficient (horizontal axis) between the extracellular vesicle fraction and the tissue expression level in Test Example 6. FIG.
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 In this specification, the expressions "contain" and "include" include the concepts of "contain", "include", "consist essentially of" and "consist only of".
　1．好酸球増多症又は気管支喘息の検査方法
　本発明は、その一態様において、好酸球増多症又は気管支喘息を検査する方法であって、（1）被検体から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質を検出する工程を含む、検査方法（本明細書において、「本発明の検査方法」と示すこともある。）に関する。以下、これについて説明する。 1. Method for testing eosinophilia or bronchial asthma In one aspect of the present invention, there is provided a method for testing eosinophilia or bronchial asthma, comprising: (1) cells of a body fluid collected from a subject; From protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G) in outer vesicles or body fluids The present invention relates to an inspection method (also referred to herein as the "inspection method of the present invention") comprising the step of detecting at least one protein selected from the group consisting of: This will be explained below.
　1-1．工程（1）
　検査対象疾患である「好酸球増多症」の種類は、特に制限されない。好酸球増多症の進行度に関する各種分類基準における全てのクラス、グレード、ステージの好酸球増多症が検査対象となり得る。好酸球増多症としては、具体的には、例えば、アレルギー性疾患、皮膚疾患、感染症、呼吸器疾患、肉芽腫性疾患、消化器疾患、内分泌疾患、自己免疫疾患、悪性腫瘍等が挙げられる。 1-1. Process (1)
The type of "eosinophilia", which is the disease to be examined, is not particularly limited. All classes, grades, and stages of eosinophilia in various classification criteria for progression of eosinophilia can be tested. Specific examples of eosinophilia include allergic diseases, skin diseases, infectious diseases, respiratory diseases, granulomatous diseases, digestive diseases, endocrine diseases, autoimmune diseases, malignant tumors, and the like. mentioned.
　アレルギー性疾患としては、例えば気管支喘息、アトピー性皮膚炎、蕁麻疹、アレルギー性鼻炎、薬剤アレルギー、アレルギー性鼻炎・結膜炎、アレルギー性気管支、アレルギー性肉芽腫性血管炎、血管性浮腫・クインケ浮腫、口腔粘膜アレルギー症候群、アレルギー性紫斑病等が挙げられる。 Examples of allergic diseases include bronchial asthma, atopic dermatitis, urticaria, allergic rhinitis, drug allergy, allergic rhinitis/conjunctivitis, allergic bronchi, allergic granulomatous vasculitis, angioedema/Quincke's edema, Oral mucosal allergy syndrome, allergic purpura, and the like.
　皮膚疾患としては、例えば尋常性天疱瘡、好酸球性膿瘡、毛包炎、湿疹、好酸球性筋膜炎、薬疹、乾癬、リンパ腫様丘疹、紅皮症等が挙げられる。 Skin diseases include, for example, pemphigus vulgaris, eosinophilic pimples, folliculitis, eczema, eosinophilic fasciitis, drug eruption, psoriasis, lymphomatous papules, and erythroderma.
　感染症としては、例えば寄生虫症、肺アスペルギルス症、結核、梅毒、トキソプラズマ症、内臓ダニ症、猩紅熱、原発性免疫不全症候群、カリニ肺炎等が挙げられる。 Infectious diseases include, for example, parasitism, pulmonary aspergillosis, tuberculosis, syphilis, toxoplasmosis, visceral tick disease, scarlet fever, primary immunodeficiency syndrome, and carinii pneumonia.
　呼吸器疾患としては、例えば気管支喘息、好酸球肺浸潤、じん肺症・珪肺症、過敏性肺臓炎、突発性敗血鉄等が挙げられる。 Examples of respiratory diseases include bronchial asthma, eosinophilic lung infiltration, pneumoconiosis/silicosis, hypersensitivity pneumonitis, and idiopathic septic iron.
　肉芽腫性疾患としては、例えば好酸球性肉芽腫症、ウェジナー肉芽腫症、サルコイドーシス、木村病等が挙げられる。 Examples of granulomatous diseases include eosinophilic granulomatosis, Wegener's granulomatosis, sarcoidosis, and Kimura disease.
　消化器疾患としては、例えば好酸球性胃腸炎、アレルギー性胃腸炎、潰瘍性大腸炎、膵臓炎、炎症性腸疾患等が挙げられる。 Gastrointestinal diseases include, for example, eosinophilic gastroenteritis, allergic gastroenteritis, ulcerative colitis, pancreatitis, and inflammatory bowel disease.
　内分泌疾患としては、例えばアジソン病、甲状腺機能亢進症等が挙げられる。 Endocrine diseases include, for example, Addison's disease and hyperthyroidism.
　自己免疫疾患としては、例えば多発性動脈炎、リウマチ性関節炎、慢性活動性肝炎、天疱瘡、サルコイドーシス、悪性関節リウマチ、結節性多発性動脈炎、カプラン症候群等が挙げられる。 Autoimmune diseases include, for example, polyarteritis, rheumatoid arthritis, chronic active hepatitis, pemphigus, sarcoidosis, malignant rheumatoid arthritis, polyarteritis nodosa, and Kaplan's syndrome.
　悪性腫瘍としては、例えば悪性リンパ腫、ホジキンリンパ腫、成人T細胞白血病、菌状息肉腫症、免疫芽球性リンパ節炎、軟部組織肉腫、がん性リンパ管症、傍悪性腫瘍症候群、重鎖病、大腸がん、肺がん等が挙げられる。 Malignant tumors include, for example, malignant lymphoma, Hodgkin's lymphoma, adult T-cell leukemia, mycosis fungoides, immunoblastic lymphadenitis, soft tissue sarcoma, carcinomatous lymphangiosis, paramalignant tumor syndrome, heavy chain disease , colon cancer, lung cancer, and the like.
　検査対象疾患である「気管支喘息」の種類は、特に制限されない。気管支喘息の進行度に関する各種分類基準における全てのクラス、グレード、ステージの気管支喘息が検査対象となり得る。また、気管支喘息のあらゆる分類カテゴリー（例えば、好酸球性喘息、アトピー性喘息等）が検査対象となり得る。 The type of "bronchial asthma" that is the disease to be tested is not particularly limited. All classes, grades, and stages of bronchial asthma in various classification criteria for progression of bronchial asthma can be tested. In addition, all classification categories of bronchial asthma (eg, eosinophilic asthma, atopic asthma, etc.) can be tested.
　被検体は、本発明の検査方法の対象生物であり、その生物種は特に制限されない。被検体の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられ、好ましくはヒトが挙げられる。 The subject is the target organism of the testing method of the present invention, and its species is not particularly limited. Species of the subject include, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans.
　被検体の状態は、特に制限されない。被検体としては、例えば好酸球増多症に罹患しているかどうか不明な検体、好酸球増多症に罹患していると既に別の方法により判定されている検体、好酸球増多症に罹患していないと既に別の方法により判定されている検体、好酸球増多症の治療中の検体、好酸球増多症の治療後の検体等が挙げられる。 The subject's condition is not particularly limited. Subjects include, for example, subjects with unknown eosinophilia, subjects already determined to have eosinophilia by another method, eosinophilia Examples include specimens that have already been determined by another method to be free of disease, specimens undergoing treatment for eosinophilia, specimens after treatment for eosinophilia, and the like.
　工程（1）における検出試料は、好ましくは体液の細胞外小胞である。 The sample to be detected in step (1) is preferably extracellular vesicles of body fluid.
　体液は、特に制限されない。体液としては、例えば全血、血清、血漿、髄液、唾液、関節液、尿、組織液（気管支肺胞洗浄液を含む）、汗、涙、喀痰、鼻汁、呼気、呼気凝縮液などが挙げられ、好ましくは全血、血清、血漿、髄液が挙げられ、より好ましくは全血、血清、血漿が挙げられる。体液は、1種単独で採用してもよいし、2種以上を組み合わせて採用してもよい。 There are no particular restrictions on body fluids. Body fluids include, for example, whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, exhaled breath, exhaled breath condensate, etc. Whole blood, serum, plasma and cerebrospinal fluid are preferred, and whole blood, serum and plasma are more preferred. One type of body fluid may be used alone, or two or more types may be used in combination.
　体液は、当業者に公知の方法で被検体から採取することができる。例えば、全血は、注射器などを用いた採血によって採取することができる。血清は、全血から血球及び特定の血液凝固因子を除去した部分であり、例えば、全血を凝固させた後の上澄みとして得ることができる。血漿は、全血から血球を除去した部分であり、例えば、全血を凝固させない条件下で遠心分離に供した際の上澄みとして得ることができる。 A body fluid can be collected from a subject by a method known to those skilled in the art. For example, whole blood can be collected by blood collection using a syringe or the like. Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as a supernatant after coagulation of whole blood. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when whole blood is subjected to centrifugation under non-clotting conditions.
　本明細書においては、全血、血清、血漿等の血液そのもの又は血液由来の試料を「血液試料」と示す。 In the present specification, blood itself such as whole blood, serum, plasma, etc., or blood-derived samples are referred to as "blood samples".
　細胞外小胞は、細胞から分泌、放出等される膜小胞である限り特に制限されない。細胞外小胞は、通常は、細胞内のタンパク質や遺伝情報（mRNA, microRNA等）を細胞外に運搬することにより、局所や全身における細胞間の情報伝達を担っている膜小胞として定義される。細胞外小胞としては、例えばエクソソーム、微小小胞体、アポトーシス小体、エクトソーム、マイクロパーティクル、分泌マイクロベシクル等が挙げられる。 Extracellular vesicles are not particularly limited as long as they are membrane vesicles that are secreted or released from cells. Extracellular vesicles are usually defined as membrane vesicles that carry intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of cells, and are responsible for local and systemic intercellular communication. be. Extracellular vesicles include, for example, exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles and the like.
　細胞外小胞は、体液から、公知の方法に従って又は準じて、精製、分離、濃縮等することができる。細胞外小胞を精製、分離、濃縮等する方法としては、例えば超遠心法（例えばペレットダウン法、スクロースクッション法、密度勾配遠心法等）、イムノアフィニティー担体を用いる方法、ゲルろ過法、フィールド・フロー分画法、FACS法等が挙げられる。また、細胞外小胞の精製、分離、濃縮等は、市販のキットを用いて行うことも可能である。これらの方法は、1種単独で採用してもよいし、2種以上を組み合わせて採用してもよい。 Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or in accordance with known methods. Methods for purifying, separating, and concentrating extracellular vesicles include, for example, ultracentrifugation (e.g., pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), methods using immunoaffinity carriers, gel filtration, field centrifugation, and the like. Flow fractionation method, FACS method and the like can be mentioned. Purification, separation, concentration and the like of extracellular vesicles can also be performed using commercially available kits. These methods may be employed singly or in combination of two or more.
　工程（1）の検出対象は、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質（本明細書において、これらをまとめて「対象タンパク質」と示すこともある。）である。 Detection targets in step (1) include protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G ) (in the present specification, these are sometimes collectively referred to as the “protein of interest”).
　タンパク質群（B）は、（B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群である。 Protein group (B) includes (B) HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelaminate A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain-containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta -dehydrogenase 11, Torsin-1A, Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase- 25, Aldehyde dehydrogenase family 3 member B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophos phatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase-activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100-A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin -10, Leucine-rich repeat and calponin homology domain-containing protein 4, and very-long-chain 3-oxoacyl-CoA reductase.
　タンパク質群（B）は、気管支喘息被検体全体の発現レベルが健常被検体の発現レベルよりも高いタンパク質である。 Protein group (B) is a protein whose expression level in all bronchial asthma subjects is higher than that in healthy subjects.
　タンパク質群（B）の中でも、好ましくはHLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1等が挙げられ、より好ましくはHLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2が挙げられる。 Among the protein group (B), preferably HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelaminate A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain-containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, etc., more preferably HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglob ulin kappa variable 2D -29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2.
　タンパク質群（C）は、（C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群である。 Protein group (C) includes (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, NucleoLysin Tia-1 IsoForm P40, NucleOlysin Tia-1 Isoform P40, NucleOlysin TIAR, DISINTEGRIN and Metalloproteinase Domain-Contening ProtEin 10, Choline T Ransport-Like Protein 2, Probable UDP-Sugar Transporter Protein SLC35A5, WD Repeat-Contening Protein 11, and Canalicular. A protein group consisting of multispecific organic anion transporter 2.
　タンパク質群（C）は、気管支喘息被検体全体の発現レベルが健常被検体の発現レベルよりも低いタンパク質である。 Protein group (C) is a protein whose expression level in all bronchial asthma subjects is lower than that in healthy subjects.
　タンパク質群（C）の中でも、好ましくはThioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26等が挙げられ、より好ましくはThioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2が挙げられる。 Among the protein group (C), preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, etc., more preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2.
　タンパク質群（D）は、（D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群である。 Protein group (D) includes (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain- containing protein 2, Microsomal glutathione S-transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin A group of proteins consisting of inhibitor heavy chain H1, long-chain-fatty-acid--CoA ligase 1, and inter-alpha-trypsin inhibitor heavy chain H2.
　タンパク質群（D）は、アトピー性喘息被検体の発現レベルが健常被検体の発現レベルよりも高いタンパク質である。 Protein group (D) is a protein whose expression level in atopic asthmatic subjects is higher than that in healthy subjects.
　タンパク質群（D）の中でも、好ましくはPutative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4等が挙げられ、より好ましくはPutative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member Aが挙げられる。 Among the protein group (D), preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol Factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, etc., more preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A.
　タンパク質群（E）は、（E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群である。 Protein group (E) includes (E) HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein , Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain- containing protein 4C, N-acetyl-D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, A group of proteins consisting of EH domain-containing protein 1, cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a.
　タンパク質群（E）は、アトピー性喘息被検体の発現レベルが健常被検体の発現レベルよりも低いタンパク質である。 Protein group (E) is a protein whose expression level in atopic asthmatic subjects is lower than that in healthy subjects.
　タンパク質群（E）の中でも、好ましくはHLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2等が挙げられ、より好ましくはHLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2が挙げられる。 Among protein group (E), preferably HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, tight junction protein ZO-2, and more preferably HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2 mentioned.
　タンパク質群（F）は、（F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群である。 Protein group (F) includes (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta -dehydrogenase 11, endoplasmic reticulum-Golgi intermediate compartment protein 1, Erlin-2, anoctamin-10, and Ras-related protein Rab-27A.
　タンパク質群（F）は、好酸球性喘息被検体の発現レベルが健常被検体の発現レベルよりも高いタンパク質である。 Protein group (F) is a protein whose expression level in eosinophilic asthmatic subjects is higher than that in healthy subjects.
　タンパク質群（F）の中でも、好ましくはGalectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein等が挙げられ、より好ましくはGalectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1が挙げられる。 Among the protein group (F), preferably Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, more preferably Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non- secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1.
　タンパク質群（G）は、（G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群である。 Protein group (G) includes (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog , Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, A group of proteins consisting of Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4, Solute carrier family 12 member 7, and Solute carrier family 43 member 3 .
　タンパク質群（G）は、好酸球性喘息被検体の発現レベルが健常被検体の発現レベルよりも低いタンパク質である。 The protein group (G) is proteins whose expression level in eosinophilic asthmatic subjects is lower than that in healthy subjects.
　タンパク質群（G）の中でも、好ましくはBone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A等が挙げられ、より好ましくはBone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilinが挙げられる。 Among the protein group (G), preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, etc., more preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin.
　タンパク質群（A）は、Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群である。 Protein group (A) is a protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil catic protein.
　タンパク質群（A）は、気管支喘息被検体の発現レベルが健常被検体の発現レベルよりも高いタンパク質であって、血中好酸球数、呼吸機能、組織病態、組織好酸球浸潤、臨床スコア、EETosis陽性好酸球数等の重症度の指標として特に好ましいタンパク質である。 Protein group (A) is a protein whose expression level in subjects with bronchial asthma is higher than that in healthy subjects, and the number of blood eosinophils, respiratory function, tissue pathology, tissue eosinophil infiltration, clinical score , EETosis-positive eosinophil count, etc., is a particularly preferable protein as an index of severity.
　タンパク質群（A）の中でも、好ましくはGalectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease等が挙げられ、特に好ましくはGalectin-10が挙げられる。 Among the protein group (A), Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, etc. are preferred, and Galectin-10 is particularly preferred.
　タンパク質群（X）は、Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群である。 Protein group (X) includes Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin-3, Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin It is a protein group consisting of heavy variable 8-61 and immunoglobulin heavy variable 9-49.
　タンパク質群（X）は、喘息の予後が悪い（鼻茸を伴う慢性副鼻腔炎を合併する喘息である）場合の発現レベルが、喘息の予後が良好である（鼻茸を伴わない慢性副鼻腔炎を合併する喘息である）場合の発現レベルよりも高いタンパク質である。 For protein group (X), the expression level of asthma with a poor prognosis (chronic sinusitis with nasal It is a protein whose expression level is higher than that in the case of comorbid asthma).
　タンパク質群（X）の中でも、好ましくはImmunoglobulin heavy variable 8-61、Eosinophil peroxidase、Immunoglobulin heavy variable 9-49、Galectin-10、Immunoglobulin heavy variable 7-43が挙げられ、より好ましくはImmunoglobulin heavy variable 8-61、Eosinophil peroxidase、Immunoglobulin heavy variable 9-49、Galectin-10が挙げられ、さらに好ましくはImmunoglobulin heavy variable 8-61が挙げられる。 Among the protein group (X), preferably Immunoglobulin heavy variable 8-61, Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, Galectin-10, Immunoglobulin heavy variable 7-43, more preferably Immunoglobulin heavy variable 8-61 , Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, and Galectin-10, more preferably Immunoglobulin heavy variable 8-61.
　タンパク質群（A）～（G）のタンパク質は、ヒトの場合であれば、後述の実施例における表１～６に示されるUniProtKBアクセッション番号によって特定されるタンパク質である。他の生物種の場合であれば、表１～６に示されるUniProtKBアクセッション番号によって特定されるタンパク質のオーソログである。 The proteins of the protein groups (A) to (G) are, in the case of humans, proteins specified by the UniProtKB accession numbers shown in Tables 1 to 6 in Examples below. For other species, it is an orthologue of the protein identified by the UniProtKB accession numbers shown in Tables 1-6.
　工程（1）における対象タンパク質の数は、1種のみでもよいが、2種以上の組み合わせであってもよい。より多く（例えば2種、5種、10種、20種、30種、40種、50種以上）の検出対象を組み合わせることにより、好酸球増多症の検査等を、より正確に行うことが可能になる。 The number of target proteins in step (1) may be one, or a combination of two or more. By combining more detection targets (e.g., 2, 5, 10, 20, 30, 40, 50 or more), eosinophilia can be tested more accurately. becomes possible.
　検出は、通常は、対象タンパク質の量又は濃度を測定することによって行われる。「濃度」とは、絶対濃度に限らず、相対濃度や、単位体積辺りの重量や、絶対濃度を知るために測定した生データなどでもよい。 Detection is usually performed by measuring the amount or concentration of the target protein. "Concentration" is not limited to absolute concentration, but may be relative concentration, weight per unit volume, or raw data measured to know absolute concentration.
　対象タンパク質を検出する方法としては、対象タンパク質の一部又は全部を特異的に検出できる方法であれば特に制限されない。検出方法としては、具体的には、例えば、対象タンパク質を構成するペプチドを検出する質量分析法、対象タンパク質を特異的に認識する抗体を用いた免疫学的測定法等を挙げることができる。なお、対象タンパク質のアミノ酸配列情報は、UniProtKBアクセッション番号を基に、EBI（http://www.ebi.ac.uk/IPI/IPIhelp.html）のデータベースで検索することにより得ることができる。 The method for detecting the target protein is not particularly limited as long as it can specifically detect part or all of the target protein. Specific examples of the detection method include mass spectrometry for detecting peptides constituting the target protein, and immunoassay using an antibody that specifically recognizes the target protein. The amino acid sequence information of the target protein can be obtained by searching the database of EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) based on the UniProtKB accession number.
　免疫学的測定法としては、免疫組織化学染色法、ELISA法、EIA法、RIA法、ウェスタンブロッティング法等を好適に例示することができる。 Immunohistochemical staining methods, ELISA methods, EIA methods, RIA methods, Western blotting methods, etc. can be suitably exemplified as immunological measurement methods.
　質量分析法とは、ペプチド試料を、イオン源を用いて気体状のイオンとし（イオン化）、分析部において、真空中で運動させ電磁気力を用いて、あるいは飛行時間差によりイオン化したペプチド試料を質量電荷比に応じて分離し、検出できる質量分析計を用いた測定方法のことをいい、イオン源を用いてイオン化する方法としては、EI法、CI法、FD法、FAB法、MALDI法、ESI法などの方法を適宜選択することができ、また、分析部において、イオン化したペプチド試料を分離する方法としては、磁場偏向型、四重極型、イオントラップ型、飛行時間（TOF）型、フーリエ変換イオンサイクロトロン共鳴型などの分離方法を適宜選択することができる。また、2以上の質量分析法を組み合わせたタンデム型質量分析（MS／MS）やトリプル四重極型質量分析を利用することができる。また、サンプルがリン酸化したペプチドを含む試料の場合、質量分析計へのサンプル導入前に、サンプルを鉄イオン固定化アフィニティークロマトグラフィー（Fe-IMAC）を用いて濃縮することができる。また、液体クロマトグラフ（LC）やHPLCにより、対象タンパク質を構成するペプチドを分離・精製してサンプルとすることができる。また、検出部やデータ処理方法も適宜選択することができる。なお、質量分析法を用いて対象タンパク質を構成するペプチドを質量分析法で検出・定量する場合、かかるペプチドと同一のアミノ酸配列からなる、濃度が既知の安定同位体で標識したペプチドを内部標準とすることができる。かかる安定同位体標識ペプチドとしては、検出する対象タンパク質を構成するペプチドにおけるアミノ酸の1個以上が、15N，13C，18O，及び2Hのいずれか1以上を含む安定同位体標識ペプチドであれば、アミノ酸の種類、位置、数などは適宜選択することができ、かかる安定同位体標識ペプチドは、安定同位元素により標識されたアミノ酸を用いてF-moc法（Amblard., et al. Methods Mol Biol.298:3-24(2005)）等の適当な手段で化学合成することができるが、iTRAQ（登録商標）試薬、ICAT（登録商標）試薬、ICPL（登録商標）試薬、NBS（登録商標）試薬などの標識試薬を用いて作製することもできる。 In mass spectrometry, a peptide sample is turned into gaseous ions (ionized) using an ion source, and the peptide sample is moved in vacuum in an analysis unit and ionized using electromagnetic force or by time-of-flight difference. A measurement method using a mass spectrometer that can separate and detect according to the ratio. Methods of ionization using an ion source include the EI method, CI method, FD method, FAB method, MALDI method, and ESI method. and other methods can be selected as appropriate, and methods for separating the ionized peptide sample in the analysis unit include magnetic field deflection, quadrupole, ion trap, time-of-flight (TOF), and Fourier transform. A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS/MS) combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used. Alternatively, if the sample contains phosphorylated peptides, the sample can be concentrated using iron ion immobilization affinity chromatography (Fe-IMAC) prior to sample introduction into the mass spectrometer. In addition, peptides constituting the target protein can be separated and purified and used as a sample by liquid chromatography (LC) or HPLC. Also, the detection unit and data processing method can be selected as appropriate. When mass spectrometry is used to detect and quantify a peptide that constitutes a target protein, a peptide that has the same amino acid sequence as the peptide and is labeled with a stable isotope of known concentration is used as an internal standard. can do. As such a stable isotope-labeled peptide, one or more of the amino acids in the peptide constituting the target protein to be detected is a stable isotope-labeled peptide containing any one or more of 15N, 13C, 18O, and 2H. can be selected as appropriate, and such stable isotope-labeled peptides are produced by the F-moc method (Amblard., et al. Methods Mol Biol. 298 : 3-24 (2005)), iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark) reagent, etc. can also be produced using a labeling reagent of
　工程（1）を含む本発明の検査方法によれば、好酸球増多症の検出指標である対象タンパク質の量及び／又は濃度を提供することができ、これにより好酸球増多症の検出、重症度の評価などを補助することができる。 According to the test method of the present invention including step (1), the amount and/or concentration of the target protein, which is a detection indicator for eosinophilia, can be provided, thereby detecting eosinophilia. It can aid in detection, assessment of severity, and the like.
　工程（1）を含む本発明の検査方法による検査結果は、好酸球増多症治療薬の治療マーカー、好酸球増多症の病態解明、好酸球増多症の予後予測、被検体の層別化、治療方法の選択（個別化医療、治療反応性）、好酸球増多症における難治化や、好酸球増多症の組織型や表現型等の鑑別等に利用し得る。 The test results obtained by the test method of the present invention including step (1) can be used as a therapeutic marker for eosinophilia therapeutic agents, elucidation of the pathology of eosinophilia, prediction of prognosis of eosinophilia, It can be used for stratification of treatment, selection of treatment method (personalized medicine, treatment response), refractory in eosinophilia, differentiation of histological type and phenotype of eosinophilia, etc. .
　1-2．工程（2）
　本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（A）、タンパク質群（B）、タンパク質群（D）、及びタンパク質群（F）からなる群より選択される少なくとも1種のタンパク質（タンパク質ABDF’）を含む場合、さらに、
（2ABDF）前記工程（1）で検出されたタンパク質ABDF’の量又は濃度がカットオフ値以上である場合に、前記被検体が好酸球増多症又は気管支喘息に罹患している、或いは高重症度の好酸球増多症又は気管支喘息に罹患している又は高重症度の好酸球増多症又は気管支喘息に将来罹患する可能性が高いと判定する工程、
を含むことが好ましい。該工程2ABDFを含む本発明の検査方法によれば、好酸球増多症又は気管支喘息を判定及び病態評価することが可能となる。 1-2. Process (2)
As one aspect of the inspection method of the present invention,
When the detected protein contains at least one protein (protein ABDF') selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F), moreover,
(2ABDF) if the amount or concentration of the protein ABDF' detected in step (1) is equal to or higher than the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future;
is preferably included. According to the testing method of the present invention including the step 2ABDF, it is possible to determine and evaluate the pathology of eosinophilia or bronchial asthma.
　本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（C）、タンパク質群（E）、及びタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質（タンパク質CEG’）を含む場合、さらに、
（2CEG）前記工程（1）で検出されたタンパク質CEG’の量又は濃度がカットオフ値以下である場合に、前記被検体が好酸球増多症又は気管支喘息に罹患している、或いは高重症度の好酸球増多症又は気管支喘息に罹患している又は高重症度の好酸球増多症又は気管支喘息に将来罹患する可能性が高いと判定する工程、
を含むことが好ましい。該工程2CEGを含む本発明の検査方法によれば、好酸球増多症を判定することが可能となる。 As one aspect of the inspection method of the present invention,
When the detected protein contains at least one protein (protein CEG') selected from the group consisting of protein group (C), protein group (E), and protein group (G),
(2CEG) if the amount or concentration of the protein CEG' detected in step (1) is less than or equal to the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future;
is preferably included. According to the testing method of the present invention including step 2CEG, it is possible to determine eosinophilia.
　検査対象疾患がアトピー性喘息である場合において、本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（D）より選択される少なくとも1種のタンパク質（タンパク質D’）を含む場合、さらに、
（2D）前記工程（1）で検出されたタンパク質D’の量又は濃度がカットオフ値以上である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
を含むことが好ましい。 In the case where the disease to be examined is atopic asthma, the examination method of the present invention comprises, as one aspect,
When the detected protein contains at least one protein (protein D') selected from the protein group (D),
(2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value;
is preferably included.
　検査対象疾患がアトピー性喘息である場合において、本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（E）より選択される少なくとも1種のタンパク質（タンパク質E’）を含む場合、さらに、
（2E）前記工程（1）で検出されたタンパク質E’の量又は濃度がカットオフ値以下である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
を含むことが好ましい。 In the case where the disease to be examined is atopic asthma, the examination method of the present invention comprises, as one aspect,
When the detected protein contains at least one protein (protein E') selected from protein group (E),
(2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value;
It is preferred to include
　検査対象疾患が好酸球性喘息である場合において、本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（F）より選択される少なくとも1種のタンパク質（タンパク質F’）を含む場合、さらに、
（2F）前記工程（1）で検出されたタンパク質F’の量又は濃度がカットオフ値以上である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
を含むことが好ましい。 In the case where the disease to be tested is eosinophilic asthma, the testing method of the present invention comprises, as one aspect,
When the detected protein contains at least one protein (protein F') selected from the protein group (F),
(2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value;
is preferably included.
　検査対象疾患が好酸球性喘息である場合において、本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（G）より選択される少なくとも1種のタンパク質（タンパク質G’）を含む場合、さらに、
（2G）前記工程（1）で検出されたタンパク質G’の量又は濃度がカットオフ値以下である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
を含むことが好ましい。 In the case where the disease to be tested is eosinophilic asthma, the testing method of the present invention comprises, as one aspect,
When the detected protein contains at least one protein (protein G') selected from the protein group (G),
(2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value;
is preferably included.
　また、高重症度の好酸球増多症の罹患の有無や将来の罹患の可能性を判定する場合には、本発明の検査方法は、一態様として、
検出したタンパク質が、タンパク質群（A）より選択される少なくとも1種のタンパク質（タンパク質A’）を含む場合、さらに、
（2A）前記工程（1）で検出されたタンパク質A’の量又は濃度がカットオフ値以上である場合に、前記被検体が高重症度の好酸球増多症又は気管支喘息に罹患している又は高重症度の好酸球増多症又は気管支喘息に将来罹患する可能性が高いと判定する工程、
を含むことが好ましい。 In addition, when determining the presence or absence of morbidity of high-severity eosinophilia and the possibility of morbidity in the future, the testing method of the present invention, as an aspect,
When the detected protein contains at least one protein (protein A') selected from protein group (A),
(2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future;
It is preferred to include
　カットオフ値は、感度、特異度、陽性的中率、陰性的中率などの観点から当業者が適宜設定することができ、例えば、対照被検体（例えば好酸球増多症に罹患していない或いは罹患している被検体、気管支喘息に罹患していない或いは罹患している被検体、アトピー性喘息に罹患していない或いは罹患している被検体、好酸球性喘息に罹患していない或いは罹患している被検体、好酸球増多症の重症度に関する指標値が低い或いは高い被検体、等）から採取された体液の細胞外小胞又は体液における対象タンパク質の量及び／又は濃度に基づいて、その都度定められた値、或いは予め定められた値とすることができる。カットオフ値は、例えば、基準となる被検体又は被検体群から採取された体液の細胞外小胞又は体液における対象タンパク質の量及び／又は濃度（被検体が複数の場合は、平均値、中央値など）の、例えば0.7～1.5倍の値とすることができる。また、受信者操作特性（Receiver Operating Characteristic, ROC）曲線の解析などに基づいた統計解析（より具体的には、Youden indexを用いた方法が例示される。）を行うことにより、設定することもできる。 The cutoff value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc. For example, control subjects (e.g., those suffering from eosinophilia subjects not suffering from or suffering from bronchial asthma, subjects not suffering from or suffering from atopic asthma, subjects not suffering from eosinophilic asthma Alternatively, the amount and/or concentration of the protein of interest in extracellular vesicles or body fluids of body fluids collected from diseased subjects, subjects with low or high index values for the severity of eosinophilia, etc. can be a value determined each time or a predetermined value based on . The cutoff value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles or body fluid of the body fluid collected from the reference subject or subject group (when the subject is multiple, the average value, median value), for example, 0.7 to 1.5 times. It can also be set by performing statistical analysis (more specifically, a method using Youden index is exemplified) based on Receiver Operating Characteristic (ROC) curve analysis, etc. can.
　工程（2）の好ましい一態様においては、被検体が好酸球増多症の治療中又は治療後の検体である場合、カットオフ値を、例えば同一検体についての過去の試料における対象タンパク質の量及び／又は濃度に基づいた値とすることにより、治療効果を判定することができる。 In a preferred embodiment of step (2), when the subject is a subject during or after treatment for eosinophilia, the cutoff value is, for example, the amount of the protein of interest in a past sample of the same subject. and/or concentration-based values can be used to determine therapeutic efficacy.
　2．好酸球増多症又は気管支喘息のより高い精度での診断
　工程（2）を含む本発明の検査方法により、被検体が好酸球増多症又は気管支喘息に罹患していると判定された場合、本発明の検査方法に、さらに好酸球増多症又は気管支喘息の医師による診断を適用する工程を組み合わせることによって、より高い精度で好酸球増多症又は気管支喘息を診断することができる。また、本発明の検査方法はより正確に好酸球増多症又は気管支喘息を検出できるので、本発明の検査方法に上記工程を組み合わせることによって、より効率的且つより正確に「好酸球増多症又は気管支喘息に罹患している」と診断できる。 2. The subject was determined to be suffering from eosinophilia or bronchial asthma by the test method of the present invention including the step (2) of diagnosing eosinophilia or bronchial asthma with higher accuracy. In some cases, eosinophilia or bronchial asthma can be diagnosed with higher accuracy by combining the testing method of the present invention with the step of applying diagnosis of eosinophilia or bronchial asthma by a doctor. can. In addition, since the testing method of the present invention can more accurately detect eosinophilia or bronchial asthma, combining the above steps with the testing method of the present invention can more efficiently and accurately detect "eosinophilia." It can be diagnosed as suffering from polymorphism or bronchial asthma.
　3．好酸球増多症又は気管支喘息の治療
　工程（2）を含む本発明の検査方法により被検体が好酸球増多症又は気管支喘息に罹患していると判定された場合は本発明の検査方法に対してさらに、或いは上記「2．好酸球増多症又は気管支喘息のより高い精度での診断」に記載の様に好酸球増多症又は気管支喘息に罹患していると診断された場合は本発明の検査方法と医師による診断を適用する工程との組合せに対してさらに、（3）好酸球増多症又は気管支喘息に罹患していると判定又は診断された被検体に対して、該疾患の治療を行う工程を行うことによって、被検体の該疾患を治療することが可能となる。また、本発明の検査方法はより正確に好酸球増多症又は気管支喘息を検出できるので、本発明の検査方法に対して、或いは本発明の検査方法と医師による診断を適用する工程との組合せに対して工程3を組み合わせることによって、好酸球増多症又は気管支喘息に罹患している被検体をより効率的に、より確実に治療できる。 3. If the subject is determined to be suffering from eosinophilia or bronchial asthma by the testing method of the present invention including the treatment step (2) for eosinophilia or bronchial asthma, the test of the present invention is performed. Diagnosed as suffering from eosinophilia or bronchial asthma further to the method or as described above in "2. Diagnosis of eosinophilia or bronchial asthma with greater accuracy". In addition to the combination of the test method of the present invention and the step of applying diagnosis by a doctor if On the other hand, by performing the step of treating the disease, it is possible to treat the disease in the subject. In addition, since the testing method of the present invention can more accurately detect eosinophilia or bronchial asthma, the testing method of the present invention or the step of applying the testing method of the present invention and a diagnosis by a doctor can be applied. By combining step 3 to the combination, subjects suffering from eosinophilia or bronchial asthma can be treated more effectively and reliably.
　好酸球増多症又は気管支喘息の治療方法は、特に制限されないが、代表的には投薬治療が挙げられる。投薬治療に用いる医薬としては、特に制限されないが、例えばアドレナリン、サルブタモール等のβ作動薬；　イプラトロピウム、チオトロピウム等の抗コリン薬；　モンテルカスト、ザフィルルカスト、ザイリュートン等のロイコトリエン修飾薬；　クロモグリク酸、ネドクロミル等の肥満細胞安定化薬；　コルチコステロイド；　オマリズマブ、メポリズマブ、レスリズマブ等の免疫調節薬（抗体医薬）；　テオフィリン等のメチルキサンチン類；　リドカイン；　ヘパリン等が挙げられる。医薬は、1種、2種、又は3種以上を組み合わせて用いることができる。 The treatment method for eosinophilia or bronchial asthma is not particularly limited, but typically includes drug treatment. Pharmaceuticals used for drug treatment are not particularly limited, but for example, β-agonists such as adrenaline and salbutamol; anticholinergics such as ipratropium and tiotropium; leukotriene modifiers such as montelukast, zafirlukast and zileuton; obesity such as cromoglycate and nedocromil cell stabilizers; corticosteroids; immunomodulators (antibody drugs) such as omalizumab, mepolizumab, reslizumab; methylxanthines such as theophylline; lidocaine; One, two, or a combination of three or more medicaments can be used.
　4．好酸球増多症又は気管支喘息の検査薬、検査キット
　本発明は、その一態様において、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、好酸球増多症又は気管支喘息の検査薬（本明細書において、「本発明の検査薬」と示すこともある。）に関する。以下、これについて説明する。 Four. Eosinophilia or bronchial asthma test agent and test kit A test agent for eosinophilia or bronchial asthma, containing at least one protein detection agent selected from the group consisting of (E), protein group (F), and protein group (G) , sometimes referred to as "the test agent of the present invention"). This will be explained below.
　タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、タンパク質群（G）、好酸球増多症又は気管支喘息等については、上記「1．好酸球増多症又は気管支喘息の検査方法」における定義と同様である。 Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
　検出剤は、対象タンパク質を特異的に検出できるものである限り特に制限されない。該検出剤としては、例えば対象タンパク質に対する抗体が挙げられる。 The detection agent is not particularly limited as long as it can specifically detect the target protein. The detection agent includes, for example, an antibody against the target protein.
　検出剤は、その機能が著しく損なわれない限りにおいて、修飾が施されていてもよい。修飾としては、例えば、標識物、例えば蛍光色素、発光物質、色素、酵素、タンパク質、放射性同位体、化学発光物質、金コロイド、ビオチン等の付加、導入等が挙げられる。 The detection agent may be modified as long as its function is not significantly impaired. Modifications include, for example, addition or introduction of labels such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, colloidal gold, biotin, and the like.
　検出剤は、任意の固相に固定化して用いることもできる。このため本発明の検査薬は、検出剤を固定化した基板（例えばプローブを固定化したマイクロアレイチップ等。別の例として、抗体を固定化したELISAプレート等）の形態として提供することができる。 The detection agent can also be used by immobilizing it on any solid phase. For this reason, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which probes are immobilized, etc. Another example is an ELISA plate on which antibodies are immobilized, etc.).
　固定化に使用される固相は、抗体等を固定化できるものであれば特に制限されることなく、例えばガラス板、ナイロンメンブレン、マイクロビーズ、シリコンチップ、キャピラリーまたはその他の基板等を挙げることができる。固相への検出剤の固定は、特に制限されない。 The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies and the like. Examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries and other substrates. can. Immobilization of the detection agent to the solid phase is not particularly limited.
　抗体は、対象タンパク質を選択的に（特異的に）認識するものであれば、特に限定されない。ここで、「選択的に（特異的に）認識する」とは、例えばウェスタンブロット法やELISA法において、対象タンパク質が特異的に検出できることを意味するが、それに限定されることなく、当業者が上記検出物が対象タンパク質に由来するものであると判断できるものであればよい。 The antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein. Here, "selectively (specifically) recognize" means that the target protein can be specifically detected in, for example, Western blotting or ELISA, but is not limited thereto. Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.
　抗体には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、またはFabフラグメントやFab発現ライブラリーによって生成されるフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。対象タンパク質のアミノ酸配列のうち少なくとも連続する、通常8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体も、本発明の抗体に含まれる。 Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or portions of the above antibodies that have antigen-binding properties, such as Fab fragments and fragments generated by Fab expression libraries. The antibody of the present invention also includes an antibody that has antigen-binding to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein.
　これらの抗体の製造方法は、すでに周知であり、本発明の抗体もこれらの常法に従って製造することができる（Current protocols in Molecular Biology , Chapter 11.12～11.13(2000)）。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従って大腸菌等で発現し精製した対象タンパク質を用いて、あるいは常法に従って当該対象タンパク質の部分アミノ酸配列を有するオリゴペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、常法に従って大腸菌等で発現し精製した対象タンパク質、あるいは対象タンパク質の部分アミノ酸配列を有するオリゴペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる（Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4～11.11）。 Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12-11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed in Escherichia coli or the like and purified according to a standard method. Then, it is possible to immunize a non-human animal such as a rabbit and obtain the serum from the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, a non-human animal such as a mouse is immunized with a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein, and spleen cells obtained by immunizing a non-human animal such as a mouse are used. It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
　抗体の作製に免疫抗原として使用される対象タンパク質は、公知の遺伝子配列情報に基づいて、DNAクローニング、各プラスミドの構築、宿主へのトランスフェクション、形質転換体の培養および培養物からのタンパク質の回収の操作により得ることができる。これらの操作は、当業者に既知の方法、あるいは文献記載の方法（Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)）などに準じて行うことができる。 The target protein used as an immunizing antigen for the production of antibodies is based on the known gene sequence information, DNA cloning, construction of each plasmid, transfection into the host, culture of the transformant, and recovery of the protein from the culture. can be obtained by the operation of These manipulations are performed according to methods known to those skilled in the art or methods described in literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). can be done.
　具体的には、対象タンパク質をコードする遺伝子が所望の宿主細胞中で発現できる組み換えDNA（発現ベクター）を作製し、これを宿主細胞に導入して形質転換し、該形質転換体を培養して、得られる培養物から、目的タンパク質を回収することによって、本発明抗体の製造のための免疫抗原としてのタンパク質を得ることができる。また対象タンパク質の部分ペプチドは、公知の遺伝子配列情報に従って、一般的な化学合成法（ペプチド合成）によって製造することもできる。 Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a target protein in a desired host cell is prepared, introduced into the host cell for transformation, and the transformant is cultured. By recovering the target protein from the resulting culture, the protein can be obtained as an immunizing antigen for producing the antibody of the present invention. A partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
　また本発明の抗体は、対象タンパク質の部分アミノ酸配列を有するオリゴペプチドを用いて調製されるものであってよい。かかる抗体の製造のために用いられるオリゴ（ポリ）ペプチドは、機能的な生物活性を有することは要しないが、対象タンパク質と同様な免疫原特性を有するものであることが望ましい。好ましくはこの免疫原特性を有し、且つ対象タンパク質のアミノ酸配列において少なくとも連続する8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるオリゴ（ポリ）ペプチドを例示することができる。 The antibody of the present invention may also be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo(poly)peptides used for the production of such antibodies need not have functional biological activity, but desirably have immunogenic properties similar to the protein of interest. An oligo(poly)peptide preferably having this immunogenic property and consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
　かかるオリゴ（ポリ）ペプチドに対する抗体の製造は、宿主に応じて種々のアジュバントを用いて免疫学的反応を高めることによって行うこともできる。限定はされないが、そのようなアジュバントには、フロイントアジュバント、水酸化アルミニウムのようなミネラルゲル、並びにリゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油乳剤、キーホールリンペットヘモシアニン及びジニトロフェノールのような表面活性物質、BCG（カルメット－ゲラン桿菌）やコリネバクテリウム-パルヴムなどのヒトアジュバントが含まれる。 Antibodies against such oligo(poly)peptides can also be produced by enhancing the immunological response using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface agents such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
　本発明の検査薬は、組成物の形態であってもよい。該組成物には、必要に応じて他の成分が含まれていてもよい。他の成分としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 The test agent of the present invention may be in the form of a composition. The composition may contain other ingredients as needed. Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.
　本発明の検査薬は、キットの形態であってもよい。該キットには、上記検出剤或いはこれを含む上記組成物のほかに、被検体の体液の細胞外小胞又は血液試料における対象タンパク質の検出に使用し得るものを含んでいてもよい。このようなものの具体例としては、各種試薬（例えば二次抗体、緩衝液等）、器具（例えば細胞外小胞の精製、分離、濃縮用器具（例えばカラム等））等が挙げられる。 The test agent of the present invention may be in the form of a kit. The kit may contain, in addition to the detection agent or the composition containing the same, those that can be used to detect the target protein in extracellular vesicles of body fluids or blood samples of a subject. Specific examples of such materials include various reagents (eg, secondary antibodies, buffer solutions, etc.), instruments (eg, extracellular vesicle purification, separation, and concentration instruments (eg, columns, etc.)), and the like.
　5．好酸球増多症又は気管支喘息の予防又は治療剤
　本発明は、その一態様において、タンパク質群（A）、タンパク質群（B）、タンパク質群（D）、並びにタンパク質群（F）からなる群より選択される少なくとも1種のタンパク質の抑制剤、並びにタンパク質群（C）、タンパク質群（E）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質の亢進剤からなる群より選択される少なくとも1種の薬剤を含有する、好酸球増多症又は気管支喘息の予防又は治療剤（本明細書において、「本発明の薬剤」と示すこともある。）に関する。以下、これについて説明する。 Five. Prophylactic or therapeutic agent for eosinophilia or bronchial asthma In one aspect of the present invention, a group consisting of protein group (A), protein group (B), protein group (D), and protein group (F) and at least one protein enhancer selected from the group consisting of protein group (C), protein group (E), and protein group (G). It relates to a prophylactic or therapeutic agent for eosinophilia or bronchial asthma (in this specification, sometimes referred to as "the drug of the present invention") containing at least one drug selected from the above. This will be explained below.
　タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、タンパク質群（G）、好酸球増多症又は気管支喘息等については、上記「1．好酸球増多症又は気管支喘息の検査方法」における定義と同様である。 Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
　抑制剤としては、例えば対象タンパク質に対する抗体が挙げられる。該抗体については、上記「4．好酸球増多症又は気管支喘息の検査薬、検査キット」で説明した抗体と同様のものを使用することができる。 Inhibitors include, for example, antibodies against target proteins. As for the antibody, the same antibody as explained in the above “4. Test agent and test kit for eosinophilia or bronchial asthma” can be used.
　抑制剤の別の例としては、対象タンパク質の発現抑制剤が挙げられる。 Another example of an inhibitor is a target protein expression inhibitor.
　対象タンパク質の発現抑制剤としては、対象タンパク質、そのmRNA、その前駆体などの発現量を抑制し得るものである限り特に制限されず、例えば対象タンパク質の遺伝子特異的small interfering RNA（siRNA）、対象タンパク質の遺伝子特異的microRNA（miRNA）、対象タンパク質の遺伝子特異的アンチセンス核酸、これらの発現ベクター；　対象タンパク質の遺伝子特異的リボザイム；　CRISPR/Casシステムによる対象タンパク質の遺伝子遺伝子編集剤などが挙げられる。 The agent for suppressing the expression of the target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, its precursor, etc. Examples include gene-specific small interfering RNA (siRNA) of the target protein, target Gene-specific microRNA (miRNA) for proteins, gene-specific antisense nucleic acids for target proteins, expression vectors for these; gene-specific ribozymes for target proteins; gene editing agents for target proteins by CRISPR/Cas system, etc.
　なお、発現抑制とは、対象タンパク質、そのmRNAなどの発現量を、例えば1/2、1/3、1/5、1/10、1/20、1/30、1/50、1/100、1/200、1/300、1/500、1/1000、1/10000以下に抑制することを意味し、これらの発現量を0とすることをも包含する。 In addition, the expression suppression means that the expression level of the target protein, its mRNA, etc. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and also includes setting the expression level to zero.
　亢進剤としては、例えば対象タンパク質の発現亢進剤が挙げられる。 Enhancers include, for example, target protein expression enhancers.
　対象タンパク質の発現亢進剤としては、対象タンパク質、そのmRNA、その前駆体などの発現量を亢進し得るものである限り特に制限されず、例えば対象タンパク質の発現ベクターなどが挙げられる。 The agent for enhancing the expression of the target protein is not particularly limited as long as it can enhance the expression level of the target protein, its mRNA, its precursor, etc. Examples include expression vectors for the target protein.
　なお、発現亢進とは、対象タンパク質、そのmRNAなどの発現量を、例えば2、3、5、10、20、30、50、100、200、300、500、1000、10000倍に亢進させることを意味する。 In addition, expression enhancement refers to enhancing the expression level of the target protein, its mRNA, etc., for example, 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, 1000, 10000 times. means.
　対象タンパク質の遺伝子siRNAは、対象タンパク質をコードする遺伝子の発現を特異的に抑制する二本鎖RNA分子である限り特に制限されない。一実施形態において、siRNAは、例えば、18塩基以上、19塩基以上、20塩基以上、又は21塩基以上の長さであることが好ましい。また、siRNAは、例えば、25塩基以下、24塩基以下、23塩基以下、又は22塩基以下の長さであることが好ましい。ここに記載するsiRNAの長さの上限値及び下限値は任意に組み合わせることが想定される。例えば、下限が18塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が19塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が20塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が21塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さの組み合わせが想定される。 The gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein. In one embodiment, the siRNA is preferably, for example, 18 bases or longer, 19 bases or longer, 20 bases or longer, or 21 bases or longer. In addition, the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. Any combination of the upper and lower limits of the siRNA length described herein is assumed. For example, a length with a lower limit of 18 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; A length with a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 21 bases with an upper limit of 25, 24, 23, or 22 bases. Combinations of lengths that are bases are envisioned.
　siRNAは、shRNA(small hairpin RNA)であっても良い。shRNAは、その一部がステムループ構造を形成するように設計することができる。例えば、shRNAは、ある領域の配列を配列aとし、配列aに対する相補鎖を配列bとすると、配列a、スペーサー、配列bの順になるようにこれらの配列が一本のRNA鎖に存在するようにし、全体で45～60塩基の長さとなるように設計することができる。配列aは、標的となる対象タンパク質をコードする塩基配列の一部の領域の配列であり、標的領域は特に限定されず、任意の領域を候補にすることが可能である。そして配列aの長さは19～25塩基、好ましくは19～21塩基である。 siRNA may be shRNA (small hairpin RNA). A portion of shRNA can be designed to form a stem-loop structure. For example, for shRNA, if the sequence of a certain region is sequence a, and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand in the order of sequence a, spacer, and sequence b. and can be designed to be 45-60 bases in length overall. Sequence a is a sequence of a partial region of the base sequence encoding the target protein of interest, and the target region is not particularly limited, and any region can be a candidate. And the length of sequence a is 19-25 bases, preferably 19-21 bases.
　対象タンパク質の遺伝子特異的siRNAは、5’又は3’末端に、付加的な塩基を有していてもよい。該付加的塩基の長さは、通常2～4塩基程度である。該付加的塩基は、DNAでもRNAでもよいが、DNAを用いると核酸の安定性を向上させることができる場合がある。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’などの配列が挙げられるが、これらに限定されるものではない。 The gene-specific siRNA of the target protein may have additional bases at the 5' or 3' end. The length of the additional bases is usually about 2-4 bases. The additional base may be either DNA or RNA, but using DNA may improve the stability of the nucleic acid. Such additional base sequences include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3 ', uuuuu-3', and the like, but are not limited to these.
　siRNAは、3'末端に突出部配列（オーバーハング）を有していてもよく、具体的には、dTdT（dTはデオキシチミジンを表わす）を付加したものが挙げられる。また、末端付加がない平滑末端（ブラントエンド）であってもよい。siRNAは、センス鎖とアンチセンス鎖が異なる塩基数であってもよく、例えば、アンチセンス鎖が3'末端及び5'末端に突出部配列（オーバーハング）を有している「asymmetrical interfering RNA（aiRNA）」を挙げることができる。典型的なaiRNAは、アンチセンス鎖が21塩基からなり、センス鎖が15塩基からなり、アンチセンス鎖の両端で各々3塩基のオーバーハング構造をとる。 The siRNA may have an overhang sequence (overhang) at the 3' end, and specifically includes those with dTdT (dT represents deoxythymidine) added. It may also be a blunt end (blunt end) without terminal addition. The siRNA may have a different number of bases in the sense strand and the antisense strand. For example, an "asymmetrical interfering RNA ( aiRNA)” can be mentioned. A typical aiRNA consists of an antisense strand of 21 nucleotides, a sense strand of 15 nucleotides, and an overhang structure of 3 nucleotides at each end of the antisense strand.
　対象タンパク質の遺伝子特異的siRNAの標的配列の位置は特に制限されるわけではないが、一実施形態において、5’-UTR及び開始コドンから約50塩基まで、並びに3’-UTR以外の領域から標的配列を選択することが望ましい。選択された標的配列の候補群について、標的以外のmRNAにおいて16-17塩基の連続した配列に相同性がないかどうかを、BLAST（http://www.ncbi.nlm.nih.gov/BLAST/）などのホモロジー検索ソフトを用いて調べ、選択した標的配列の特異性を確認することが好ましい。特異性が確認された標的配列について、AA（もしくはNA）以降の19－21塩基にTTもしくはUUの3’末端オーバーハングを有するセンス鎖と、該19－21塩基に相補的な配列及びTTもしくはUUの3’末端オーバーハングを有するアンチセンス鎖とからなる2本鎖RNAをsiRNAとして設計してもよい。また、siRNAの前駆体であるshRNAは、ループ構造を形成しうる任意のリンカー配列（例えば、5-25塩基程度）を適宜選択し、上記センス鎖とアンチセンス鎖とを該リンカー配列を介して連結することにより設計することができる。 The position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, the target sequence is from the 5'-UTR and the start codon to about 50 bases and from a region other than the 3'-UTR. Sequence selection is preferred. BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) to confirm the specificity of the selected target sequence. For the target sequence whose specificity has been confirmed, a sense strand having a TT or UU 3' overhang at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a UU 3'-terminal overhang may be designed as an siRNA. In addition, for shRNA, which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5 to 25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and antisense strand are connected via the linker sequence. It can be designed by concatenating.
　siRNA及び／又はshRNAの配列は、種々のwebサイト上に無料で提供される検索ソフトを用いて検索が可能である。このようなサイトとしては、例えば、以下を挙げることができる。
Ambionが提供するsiRNA Target Finder（http://www.ambion.com/jp/techlib/misc/siRNA_finder.html）pSilencer（登録商標）Expression Vector用インサートデザインツール（http://www.ambion.com/jp/techlib/misc/psilencer_converter.html）RNAi Codexが提供するGeneSeer（http://codex.cshl.edu/scripts/newsearchhairpin.cgi）。 siRNA and/or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include the following.
siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp/techlib/misc/psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
　siRNAは、mRNA上の標的配列のセンス鎖及びアンチセンス鎖をDNA/RNA自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約90～約95℃で約1分程度変性させた後、約30～約70℃で約1～約8時間アニーリングさせることにより調製することができる。また、siRNAの前駆体となるshRNAを合成し、これを、RNA切断タンパク質ダイサー（dicer）を用いて切断することにより調製することもできる。 siRNA is prepared by synthesizing the sense strand and antisense strand of the target sequence on mRNA with an automatic DNA/RNA synthesizer, and denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute. It can be prepared by annealing at about 30 to about 70° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with the RNA-cleaving protein dicer.
　対象タンパク質の遺伝子特異的miRNAは、対象タンパク質をコードする遺伝子の翻訳を阻害する限り任意である。例えば、miRNAは、siRNAのように標的mRNAを切断するのではなく、標的の3’非翻訳領域（UTR）に対合してその翻訳を阻害してもよい。miRNAは、pri-miRNA（primary miRNA）、pre-miRNA（precursor miRNA）、及び成熟miRNAのいずれでもよい。miRNAの長さは特に制限されず、pri-miRNAの長さは通常数百～数千塩基であり、pre-miRNAの長さは通常50～80塩基であり、成熟miRNAの長さは通常18～30塩基である。一実施形態において、対象タンパク質の遺伝子特異的miRNAは、好ましくはpre-miRNA又は成熟miRNAであり、より好ましくは成熟miRNAである。このような対象タンパク質の遺伝子特異的miRNAは、公知の手法で合成してもよく、合成RNAを提供する会社から購入してもよい。 The gene-specific miRNA for the target protein is optional as long as it inhibits translation of the gene encoding the target protein. For example, miRNAs may bind to the 3' untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA as siRNAs do. miRNA may be pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), or mature miRNA. The length of miRNA is not particularly limited, and the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50-80 bases, and the length of mature miRNA is usually 18 bases. ~30 bases. In one embodiment, the gene-specific miRNA of the protein of interest is preferably pre-miRNA or mature miRNA, more preferably mature miRNA. Such gene-specific miRNAs of target proteins may be synthesized by known methods or purchased from companies that provide synthetic RNAs.
　対象タンパク質の遺伝子特異的アンチセンス核酸とは、対象タンパク質をコードする遺伝子のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列又はその一部を含む核酸であって、該mRNAと特異的かつ安定した二重鎖を形成して結合することにより、対象タンパク質合成を抑制する機能を有する核酸である。アンチセンス核酸はDNA、RNA、DNA/RNAキメラのいずれでもよい。アンチセンス核酸がDNAの場合、標的RNAとアンチセンスDNAとによって形成されるRNA:DNAハイブリッドは、内在性リボヌクレアーゼH（RNase H）に認識されて標的RNAの選択的な分解を引き起こす。したがって、RNase Hによる分解を指向するアンチセンスDNAの場合、標的配列は、mRNA中の配列だけでなく、対象タンパク質の遺伝子の初期翻訳産物におけるイントロン領域の配列であってもよい。イントロン配列は、ゲノム配列と、対象タンパク質の遺伝子のcDNA塩基配列とをBLAST、FASTAなどのホモロジー検索プログラムを用いて比較することにより、決定することができる。 A gene-specific antisense nucleic acid for a target protein is a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the gene encoding the target protein, or a part thereof, and is specific to the mRNA. It is a nucleic acid that has the function of inhibiting target protein synthesis by forming and binding to a stable double strand. Antisense nucleic acids can be DNA, RNA, or DNA/RNA chimeras. When the antisense nucleic acid is DNA, an RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) to cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of interest protein. The intron sequence can be determined by comparing the genomic sequence and the cDNA nucleotide sequence of the gene of the target protein using homology search programs such as BLAST and FASTA.
　対象タンパク質の遺伝子特異的アンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果として対象タンパク質への翻訳が阻害されるものであればその長さは制限されない。対象タンパク質の遺伝子特異的アンチセンス核酸は、対象タンパク質をコードするmRNAの全配列であっても部分配列であってもよい。合成の容易さや抗原性、細胞内移行性の問題などを考慮すれば、約10～約40塩基、特に約15～約30塩基からなるオリゴヌクレオチドが好ましいが、これらに限定されるものではない。より具体的には、対象タンパク質の遺伝子の5’端ヘアピンループ、5’端非翻訳領域、翻訳開始コドン、タンパク質コード領域、ORF翻訳終止コドン、3’端非翻訳領域、3’端パリンドローム領域又は3’端ヘアピンループなどをアンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されるものではない。 The target region of the gene-specific antisense nucleic acid of the target protein is not limited in length as long as the hybridization of the antisense nucleic acid results in inhibition of translation into the target protein. A gene-specific antisense nucleic acid for a protein of interest may be the entire sequence or a partial sequence of the mRNA encoding the protein of interest. Oligonucleotides of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred in consideration of ease of synthesis, antigenicity, intracellular translocation, etc., but are not limited to these. More specifically, 5' terminal hairpin loop, 5' terminal untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3' terminal untranslated region, 3' terminal palindromic region of gene of target protein Alternatively, the 3' end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
　対象タンパク質の遺伝子特異的アンチセンス核酸は、対象タンパク質の遺伝子のmRNAや初期転写産物とハイブリダイズしてタンパク質への翻訳を阻害するだけでなく、二本鎖DNAであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、RNAへの転写を阻害し得るもの（アンチジーン（antigene））であってもよい。 The gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA or initial transcript of the target protein gene to inhibit translation into protein, but also binds to these genes, which are double-stranded DNA. It may be one that can form a triplex and inhibit transcription to RNA (antigene).
　上記した対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、及び対象タンパク質の遺伝子特異的アンチセンス核酸を構成するヌクレオチド分子は、安定性（化学的及び／又は対酵素）や比活性（RNAとの親和性）を向上させるために、種々の化学修飾を含んでもよい。例えば、ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンス核酸を構成する各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（phosphorothioate; PS)、メチルホスホネート（methylphosphonate）、ホスホロジチオネート（phosphorodithioate）などの化学修飾リン酸残基に置換することができる。また、各ヌクレオチドの糖（リボース）の2’位の水酸基を、-OR（R=CH3（2’-O-Me）、CH2CH2OCH3（2’-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、又はCH2CH2CNなど）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などを施してもよい。また、siRNAやmiRNAを構成するヌクレオチド分子の一部は、天然型のDNAに置換されていてもよい。 The nucleotide molecules that make up the gene-specific siRNA of the protein of interest, the gene-specific miRNA of the protein of interest, and the gene-specific antisense nucleic acid of the protein of interest described above have stability (chemical and/or enzymatic) and specific activity ( Various chemical modifications may be included in order to improve affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphate residue (phosphate) of each nucleotide that constitutes the antisense nucleic acid is replaced with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithioate. can be substituted with chemically modified phosphate residues such as phosphorodithioates. In addition, the hydroxyl group at the 2' position of the sugar (ribose) of each nucleotide is replaced by -OR (R=CH 3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH 2 CH2NHC ( NH) NH2 , CH2CONHCH3 , or CH2CH2CN ) . Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or cationic functional group to the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. may be applied. In addition, part of the nucleotide molecules that constitute siRNA or miRNA may be replaced with natural DNA.
　対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、及び対象タンパク質の遺伝子特異的アンチセンス核酸などは、対象タンパク質の遺伝子のcDNA配列もしくはゲノミックDNA配列に基づいてmRNAもしくは初期転写産物の標的配列を決定し、市販のDNA/RNA自動合成機を用いて、これに相補的な配列を合成することにより調製することができる。また、上記した各種修飾を含むアンチセンス核酸も、いずれも公知の手法により、化学的に合成することができる。 Gene-specific siRNAs for proteins of interest, gene-specific miRNAs for proteins of interest, and gene-specific antisense nucleic acids for proteins of interest, etc., target mRNA or early transcripts based on the cDNA or genomic DNA sequence of the gene for the protein of interest. It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA/RNA automatic synthesizer. In addition, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known techniques.
　対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、対象タンパク質の遺伝子特異的アンチセンス核酸の発現ベクター、対象タンパク質の発現ベクター等については、対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、対象タンパク質の遺伝子特異的アンチセンス核酸、対象タンパク質のmRNA等が発現可能な状態で組み込まれている限りにおいて特に限定されない。典型的には、該発現ベクターは、プロモーター配列、及び対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、対象タンパク質の遺伝子特異的アンチセンス核酸、又は対象タンパク質のコード配列（必要に応じて、さらに転写終結シグナル配列）を含むポリヌクレオチド、必要に応じて他の配列を含む。プロモーターは、特に制限されず、例えばCMVプロモーター、EF1プロモーター、SV40プロモーター、MSCVプロモーター、hTERTプロモーター、βアクチンプロモーター、CAGプロモーターなどのRNA polymerase II（polII）系プロモーター；　マウス及びヒトのU6-snRNAプロモーター、ヒトH1-RNase P RNAプロモーター、ヒトバリン-tRNAプロモーターなどのRNA polymerase III（polIII）系プロモーターなどが挙げられる。他の配列としては、特に制限されず、発現ベクターが含み得る公知の配列を各種採用することができる。このような配列の一例としては、例えば複製起点、薬剤耐性遺伝子などが挙げられる。また、薬剤耐性遺伝子の種類及びベクターの種類は上述のものを例示できる。 Gene-specific siRNA of target protein, gene-specific miRNA of target protein, expression vector of gene-specific antisense nucleic acid of target protein, expression vector of target protein, gene-specific siRNA of target protein, gene of target protein There is no particular limitation as long as the specific miRNA, the gene-specific antisense nucleic acid of the target protein, the mRNA of the target protein, etc. are incorporated in an expressible state. Typically, the expression vector comprises a promoter sequence and a gene-specific siRNA for the protein of interest, a gene-specific miRNA for the protein of interest, a gene-specific antisense nucleic acid for the protein of interest, or a coding sequence for the protein of interest (optionally and a transcription termination signal sequence), optionally other sequences. The promoter is not particularly limited, for example, RNA polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter; mouse and human U6-snRNA promoters; Examples thereof include RNA polymerase III (polIII) promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter. Other sequences are not particularly limited, and various known sequences that can be contained in an expression vector can be employed. Examples of such sequences include origins of replication, drug resistance genes, and the like. Moreover, the types of drug-resistant genes and the types of vectors can be exemplified by those described above.
　対象タンパク質の遺伝子発現抑制剤の別の例としては、対象タンパク質の遺伝子特異的リボザイムなどが挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するRNAを意味するが、本願では配列特異的な核酸切断活性を有する限りDNAをも包含する。リボザイム核酸として最も汎用性の高いものは、ウイロイドやウイルソイドなどの感染性RNAに見られるセルフスプライシングRNAがあり、ハンマーヘッド型やヘアピン型などが知られている。ハンマーヘッド型は約40塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約10塩基程度）をmRNAの所望の切断部位と相補的な配列にすることにより、標的mRNAのみを特異的に切断することが可能である。このタイプのリボザイム核酸は、RNAのみを基質とするので、ゲノムDNAを攻撃することがないという利点を有する。対象タンパク質の遺伝子のmRNAが自身で二本鎖構造をとる場合には、RNAヘリカーゼと特異的に結合し得るウイルス核酸由来のRNAモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)］。さらに、リボザイムを、それをコードするDNAを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、tRNAを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる［Nucleic Acids Res., 29(13): 2780-2788 (2001)］。 Another example of a gene expression inhibitor for a target protein is a gene-specific ribozyme for the target protein. The term "ribozyme" in the narrow sense means RNA having enzymatic activity to cleave nucleic acid, but in the present application, it also includes DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acids are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exhibits enzymatic activity at about 40 bases, and a few bases at each end (about 10 bases in total) adjacent to the part that forms the hammerhead structure are linked to the sequence complementary to the desired cleavage site of the mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the advantage that it does not attack genomic DNA because it uses only RNA as a substrate. When the mRNA of the gene of the target protein has a double-stranded structure by itself, the target sequence is converted to a single strand by using a hybrid ribozyme that ligates an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase. [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when a ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which a tRNA-modified sequence is further ligated in order to promote translocation of the transcript into the cytoplasm. [Nucleic Acids Res., 29(13): 2780-2788 (2001)].
　本発明の薬剤の適用対象は特に限定されず、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物などが挙げられる。 The application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. .
　本発明の薬剤の形態は、特に限定されず、本発明の薬剤の用途に応じて、各用途において通常使用される形態をとることができる。 The form of the drug of the present invention is not particularly limited, and can take any form commonly used for each application depending on the use of the drug of the present invention.
　形態としては、用途が医薬、健康増進剤、栄養補助剤（サプリメントなど）などである場合は、例えば錠剤（口腔内側崩壊錠、咀嚼可能錠、発泡錠、トローチ剤、ゼリー状ドロップ剤などを含む）、丸剤、顆粒剤、細粒剤、散剤、硬カプセル剤、軟カプセル剤、ドライシロップ剤、液剤（ドリンク剤、懸濁剤、シロップ剤を含む）、ゼリー剤などの経口摂取に適した製剤形態（経口製剤形態）、点鼻剤、吸入剤、肛門坐剤、挿入剤、浣腸剤、ゼリー剤、注射剤、貼付剤、ローション剤、クリーム剤などの非経口摂取に適した製剤形態（非経口製剤形態）が挙げられる。 Forms include, for example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops, etc.) when the application is pharmaceuticals, health-promoting agents, nutritional supplements (supplements, etc.), etc. ), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly preparations suitable for oral intake forms (oral formulations), formulations suitable for parenteral intake such as nasal drops, inhalants, rectal suppositories, inserts, enemas, jelly, injections, patches, lotions, cream oral dosage forms).
　形態としては、用途が食品組成物の場合は、液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、豆乳、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキーなどが挙げられる。 As for the form, when the application is a food composition, liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants , cake mixes, powdered or liquid dairy products, breads, cookies, etc.
　形態としては、用途が口腔用組成物である場合は、例えば液体（溶液、乳液、懸濁液など）、半固体（ゲル、クリーム、ペーストなど）、固体（錠剤、粒子状剤、カプセル剤、フィルム剤、混練物、溶融固体、ロウ状固体、弾性固体など）などの任意の形態、より具体的には、歯磨剤（練歯磨、液体歯磨、液状歯磨、粉歯磨など）、洗口剤、塗布剤、貼付剤、口中清涼剤、食品（例えば、チューインガム、錠菓、キャンディ、グミ、フィルム、トローチなど）などが挙げられる。 As for the form, when the application is an oral composition, for example, liquid (solution, milky lotion, suspension, etc.), semisolid (gel, cream, paste, etc.), solid (tablet, granule, capsule, Film agent, kneaded product, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid dentifrice, liquid dentifrice, toothpaste, etc.), mouthwash, Coating agents, patches, mouth fresheners, foods (eg, chewing gum, tablet candy, candy, gummies, films, lozenges, etc.) and the like.
　本発明の薬剤は、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、例えば医薬、食品組成物、口腔用組成物、健康増進剤、栄養補助剤（サプリメントなど）などに配合され得る成分である限り特に限定されるものではないが、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤などが挙げられる。 The drug of the present invention may further contain other ingredients as necessary. Other ingredients are not particularly limited as long as they are ingredients that can be blended in pharmaceuticals, food compositions, oral compositions, health-promoting agents, nutritional supplements (supplements, etc.). , carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, chelating agents and the like.
　本発明の薬剤の対象タンパク質の抑制剤及び亢進剤の合計含有量は、抑制剤及び亢進剤の種類、用途、使用態様、適用対象、適用対象の状態などに左右されるものであり、限定はされないが、例えば0.0001～100重量％、好ましくは0.001～50重量％とすることができる。 The total content of the suppressor and enhancer for the target protein of the drug of the present invention depends on the type of suppressor and enhancer, application, mode of use, application target, condition of application target, etc., and the limitation is However, it can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.
　本発明の組成物の適用（例えば、投与、摂取、接種など）量は、薬効を発現する有効量であれば特に限定されず、通常は、有効成分の重量として、一般に一日あたり0.1～1000 mg/kg体重である。上記投与量は1日1回又は2～3回に分けて投与するのが好ましく、年齢、病態、症状により適宜増減することもできる。 The amount of application (for example, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits efficacy. mg/kg body weight. The above dosage is preferably administered once or divided into 2 to 3 times a day, and can be adjusted appropriately according to age, condition and symptoms.
　6．好酸球増多症又は気管支喘息の予防又は治療剤の有効成分のスクリーニング方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の予防又は治療剤の有効成分（又はその候補物質）のスクリーニング方法（本明細書において、「本発明の有効成分スクリーニング方法」と示すこともある。）に関する。以下、これについて説明する。 6. A screening method for an active ingredient of a preventive or therapeutic agent for eosinophilia or bronchial asthma , Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), and Protein Group (G) A screening method for an active ingredient (or a candidate substance thereof) of a prophylactic or therapeutic agent for eosinophilia or bronchial asthma, using the amount or concentration of at least one protein as an indicator (herein referred to as "the (also referred to as “active ingredient screening method”). This will be explained below.
　体液、細胞外小胞、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、タンパク質群（G）、好酸球増多症又は気管支喘息、対象タンパク質の量又は濃度の測定等については、上記「1．好酸球増多症又は気管支喘息の検査方法」における定義と同様である。 body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), protein group (G), Hyperacidophilia or bronchial asthma, measurement of the amount or concentration of the target protein, etc. are defined in the above "1. Test method for eosinophilia or bronchial asthma".
　動物の生物種は特に制限されない。動物の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられる。 There are no particular restrictions on animal species. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
　被検物質としては、天然に存在する化合物又は人工に作られた化合物を問わず広く使用することができる。また、精製された化合物に限らず、多種の化合物を混合した組成物や、動植物の抽出液も使用することができる。化合物には、低分子化合物に限らず、タンパク質、核酸、多糖類等の高分子化合物も包含される。 A wide range of test substances can be used, regardless of whether they are naturally occurring compounds or artificially created compounds. In addition, not only purified compounds, but also compositions in which various compounds are mixed, and extracts of animals and plants can be used. Compounds include not only low-molecular-weight compounds, but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.
　本発明の有効成分スクリーニング方法は、より具体的には、タンパク質群（A）、タンパク質群（B）、タンパク質群（D）、並びにタンパク質群（F）からなる群より選択される少なくとも1種のタンパク質に関する前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞又は血液試料における対応タンパク質の量又は濃度よりも低い場合に、前記被検物質を好酸球増多症又は気管支喘息の予防又は治療剤の有効成分として選択する工程、並びに
タンパク質群（C）、タンパク質群（E）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質に関する前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞又は血液試料における対応タンパク質の量又は濃度よりも高い場合に、前記被検物質を好酸球増多症又は気管支喘息の予防又は治療剤の有効成分として選択する工程
からなる群より選択される少なくとも1種の工程を含む。 More specifically, the active ingredient screening method of the present invention comprises at least one selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F). A test substance is eosinophilic if the value of the indicator for the protein is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance. A step of selecting as an active ingredient of a prophylactic or therapeutic agent for pleocytosis or bronchial asthma, and at least one selected from the group consisting of protein group (C), protein group (E), and protein group (G) The test substance is eosinophilic if the value of the indicator for the protein is higher than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance. It comprises at least one step selected from the group consisting of the steps of selecting an active ingredient for a prophylactic or therapeutic agent for bloating or bronchial asthma.
　対応タンパク質とは、指標としている対象タンパク質と同じタンパク質を意味する。 A corresponding protein means the same protein as the target protein used as an index.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 "Low" means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 "High" means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
　7．好酸球増多症又は気管支喘息の誘発性又は増悪性の評価方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液の細胞外小胞又は血液試料における、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の誘発性又は増悪性の評価方法（本明細書において、「本発明の毒性評価方法」と示すこともある。）に関する。以下、これについて説明する。 7. In one aspect of the present invention, a method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma is characterized by: At least selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G) A method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma using the amount or concentration of one kind of protein as an index (in the present specification, it may be referred to as the "toxicity evaluation method of the present invention") .) regarding. This will be explained below.
　体液、細胞外小胞、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、タンパク質群（G）、好酸球増多症又は気管支喘息、対象タンパク質の量又は濃度の測定、動物の生物種、被検物質等については、上記「1．好酸球増多症の検査方法」及び「6．好酸球増多症又は気管支喘息の予防又は治療剤の有効成分のスクリーニング方法」における定義と同様である。 body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), protein group (G), For hyperacidophilia or bronchial asthma, measurement of the amount or concentration of the target protein, animal species, test substance, etc., see "1. Test method for eosinophilia" and "6. Eosinophilia" above. Screening method for active ingredients of prophylactic or therapeutic agents for pleurisy or bronchial asthma".
　本発明の毒性評価方法は、より具体的には、タンパク質群（A）、タンパク質群（B）、タンパク質群（D）、並びにタンパク質群（F）からなる群より選択される少なくとも1種のタンパク質に関する前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞又は血液試料における対応タンパク質の量又は濃度よりも高い場合に、前記被検物質を好酸球増多症又は気管支喘息の誘発性又は増悪性があると判定する工程、及び
タンパク質群（C）、タンパク質群（E）、並びにタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質に関する前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞又は血液試料における対応タンパク質の量又は濃度よりも低い場合に、前記被検物質を好酸球増多症又は気管支喘息の誘発性又は増悪性があると判定する工程
からなる群より選択される少なくとも1種の工程を含む。 More specifically, the toxicity evaluation method of the present invention includes at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F). is greater than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance Determining proliferative disease or bronchial asthma provocation or exacerbation, and at least one protein selected from the group consisting of protein group (C), protein group (E), and protein group (G) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance It comprises at least one step selected from the group consisting of the steps of judging proliferative or exacerbating polycythemia or bronchial asthma.
　対応タンパク質とは、指標としている対象タンパク質と同じタンパク質を意味する。 A corresponding protein means the same protein as the target protein used as an index.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 "High" means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 "Low" means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.
　試験例1．細胞外小胞画分の調製
　気管支喘息であると診断されたヒト被検体（8名（好酸球性喘息4名、アトピー性喘息4名））それぞれの血清、及び健常ヒト被検体（4名）それぞれの血清から細胞外小胞画分を調製した。細胞外小胞画分の調製は、各検体の血清の容量を揃えて、細胞外小胞調製キット（MagCaptur（商標） エクソソームアイソレーションキット PS、富士フイルム和光純薬社製）を用いて行った。 Test example 1. Preparation of extracellular vesicle fraction Serum from 8 human subjects diagnosed with bronchial asthma (4 with eosinophilic asthma, 4 with atopic asthma) and 4 healthy human subjects ) Extracellular vesicle fractions were prepared from each serum. The extracellular vesicle fraction was prepared using an extracellular vesicle preparation kit (MagCaptur™ Exosome Isolation Kit PS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with the same volume of serum for each sample. Ta.
　続いて、得られた細胞外小胞画分について、細胞外小胞の粒子数及び粒子径を測定した。具体的には、ナノサイト（日本カンタム・デザイン株式会社、Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025）を使用して測定した。これは、粒子径ごとのブラウン運動速度の違いをもとに解析するものであり、画面に映る散乱光１つ１つの動きを追尾（トラッキング）し、各々の移動速度（拡散係数）から液中における粒子径（流体力学径）を算出することができる。 Subsequently, the extracellular vesicle particle count and particle size were measured for the obtained extracellular vesicle fraction. Specifically, it was measured using Nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size. The particle size (hydrodynamic diameter) in can be calculated.
　さらに、細胞外小胞を免疫電子顕微鏡法によって観察した。具体的には、次のようにして行った。固定済み細胞外小胞溶液を5～8μLずつグリッドへ載せ15分静置して、グリッドのフォルムバールへ細胞外小胞を定着させた。PBSで3回洗浄した後、ブロッキング反応（1%BSA/PBS、10分）を行い、続いて一次抗体反応（Invitrogen AHS0902  Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2/57、100倍希釈、2時間半、室温）を行った。PBSで6回洗浄した後、二次抗体反応（CRL EMGMHL10 Anti-IgG (H＋L), Mouse, Goat-Poly, Gold 10 nm, EM、200倍希釈、1時間、室温）を行った。PBSで6回洗浄した後、1%GA/PBSで15分間処理し、さらにPBSで3回洗浄した。水洗し、0.5% UA（酢酸ウラニル）で処理して、乾燥させて、電子顕微鏡で観察した。 In addition, extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was carried out as follows. 5 to 8 μL of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to allow the extracellular vesicles to settle on the formvar of the grid. After washing with PBS three times, a blocking reaction (1% BSA/PBS, 10 minutes) was performed, followed by a primary antibody reaction (Invitrogen AHS0902 Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2/57, 100-fold dilution, 2.5 hours, room temperature). After washing with PBS six times, secondary antibody reaction (CRL EMGMHL10 Anti-IgG (H+L), Mouse, Goat-Poly, Gold 10 nm, EM, 200-fold dilution, 1 hour, room temperature) was performed. After washing 6 times with PBS, the cells were treated with 1% GA/PBS for 15 minutes, and washed 3 times with PBS. It was washed with water, treated with 0.5% UA (uranyl acetate), dried, and observed with an electron microscope.
　またさらに、細胞外小胞画分について、エクソソームマーカーに対する抗体（抗Flotillin-1抗体、抗CD9抗体、及び抗CD81抗体）を用いて、ウェスタンブロットした。 Furthermore, extracellular vesicle fractions were Western blotted using antibodies against exosome markers (anti-Flotillin-1 antibody, anti-CD9 antibody, and anti-CD81 antibody).
　上記解析の結果、得られた画分にはエクソソームが存在していることが確認できた。 As a result of the above analysis, it was confirmed that exosomes were present in the obtained fraction.
　試験例2．プロテオミクス解析（DIAプロテオーム解析）
　試験例1で得られた細胞外小胞画分中のタンパク質の定量を、DIAプロテオーム解析により行った。具体的には以下のようにして行った。 Test example 2. Proteomics analysis (DIA proteome analysis)
Proteins in the extracellular vesicle fraction obtained in Test Example 1 were quantified by DIA proteome analysis. Specifically, it was carried out as follows.
　10%デオキシコール酸ナトリウムを含む1M Tris-HCl pH8.5をエクソソーム溶出液100μlに添加し、BIORUPTOR UCW-201（SonicBio Corporation）を用いて "High "で10分間ソニケートした。サンプルを10mMジチオスレイトールで50℃で30分間処理した後、30mMヨードアセトアミドを用いて室温で30分間暗所でアルキル化した。アルキル化したサンプルを室温で60mMシステインで10分間インキュベートし、次いで50mM重炭酸アンモニウムで200μL希釈し、37℃で一晩500 ng Trypsin/Lys-C Mixで消化した。消化したペプチドを沈殿させ、5％トリフルオロ酢酸30μLで酸性化して洗浄剤を除去し、サンプルを17,400×gで15分間遠心分離した。上清をC18-StageTipを用いて脱塩し、遠心エバポレーターを用いて乾燥し、乾燥したペプチドを3％ACNおよび0.1％FAに溶解した。 　1M Tris-HCl pH8.5 containing 10% sodium deoxycholate was added to 100 μl of exosome eluate and sonicated for 10 minutes at "High" using BIORUPTOR UCW-201 (SonicBio Corporation). Samples were treated with 10 mM dithiothreitol for 30 min at 50° C. and then alkylated with 30 mM iodoacetamide for 30 min at room temperature in the dark. Alkylated samples were incubated with 60 mM cysteine for 10 minutes at room temperature, then diluted to 200 μL with 50 mM ammonium bicarbonate and digested with 500 ng Trypsin/Lys-C Mix overnight at 37°C. Digested peptides were precipitated, acidified with 30 μL of 5% trifluoroacetic acid to remove detergent, and samples were centrifuged at 17,400×g for 15 minutes. The supernatant was desalted using a C18-StageTip, dried using a centrifugal evaporator, and the dried peptide was dissolved in 3% ACN and 0.1% FA.
　ショットガンプロテオーム解析の前処理は、以前に報告されているように行った(PMID: 31779068)。ペプチドは、C18コアシェル粒子（CAPCELL CORE MP 2.7μm、160Å材料；大阪曹達株式会社）を充填した75μm×20cmのPicoFritエミッター（New Objective）に50℃で直接注入し、UltiMate 3000 RSLCnano LCシステム（Thermo Fisher Scientific）を用いて100nl/minの流速で90分間のグラジエントで分離した。カラムから溶出したペプチドは、Q Exactive HF-X (Thermo Fisher Scientific)を用いてオーバーラップウィンドウDIA-MS (PMID: 30671891, PMID: 31779068)で分析した。MS1 スペクトルは、3e6 の自動ゲイン制御ターゲットと最大注入時間 55 を設定するために、495-785 m/z の範囲で 30,000 の分解能で収集された。MS2は、自動利得制御目標を3e6、最大入射時間を "auto"、ステップ付き正規化衝突エネルギーを24, 26, 28%に設定するために、15,000の分解能で200m/z以上のスペクトルを収集した。MS2 のアイソレーション幅は 4 m/z に設定し、500-780 m/z の重なり合うウィンドウパターンは、Skyline (PMID: 20147306) で最適化されたウィンドウ配置を使用した。 　Pretreatment for shotgun proteome analysis was performed as previously reported (PMID: 31779068). Peptides were injected directly into a 75 μm × 20 cm PicoFrit emitter (New Objective) packed with C18 core-shell particles (CAPCELL CORE MP 2.7 μm, 160 Å material; Osaka Soda Co., Ltd.) at 50 °C and analyzed using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) at a flow rate of 100 nl/min with a gradient of 90 minutes. Peptides eluted from the column were analyzed by overlapping window DIA-MS (PMID: 30671891, PMID: 31779068) using Q Exactive HF-X (Thermo Fisher Scientific). MS1 spectra were collected at a resolution of 30,000 over the range 495-785 m/z to set an automatic gain control target of 3e6 and a maximum injection time of 55. MS2 collected spectra over 200 m/z at a resolution of 15,000 to set the automatic gain control target to 3e6, the maximum injection time to "auto", and the stepped normalized collision energies to 24, 26, and 28%. . The MS2 isolation width was set to 4 m/z and the 500-780 m/z overlapping window pattern used the optimized window placement in Skyline (PMID: 20147306).
　MSファイルは、Scaffold DIA (Proteome Software, Inc., Portland, OR)を用いてマウススペクトルライブラリに対して検索した。マウスのスペクトルライブラリは、Prosit (PMID: 32214105, PMID: 31133760) によるヒトタンパク質配列データベース (UniProt id UP000005640, reviewed, canonical) から生成した。Scaffold DIAの検索パラメータは、実験データ検索酵素、トリプシン、最大欠失開裂部位、1、前駆体質量許容値、8ppm、フラグメント質量許容値、10ppm、静的修飾、システインカルバミドメチル化とした。タンパク質同定閾値は、ペプチドおよびタンパク質の偽発見率の両方を1％未満に設定した。ペプチドの定量は、Scaffold DIAのEncyclopeDIAアルゴリズム(PMID: 30510204)で計算した。各ペプチドについて、最高品質のフラグメントイオンを4つ選択して定量した。蛋白質の定量は、ペプチドの定量を合計したものから推定した。 MS files were searched against the mouse spectral library using Scaffold DIA (Proteome Software, Inc., Portland, OR). Mouse spectral libraries were generated from the human protein sequence database (UniProt id UP000005640, reviewed, canonical) by Prosit (PMID: 32214105, PMID: 31133760). The search parameters for Scaffold DIA were: experimental data search enzyme, trypsin, maximum deletion cleavage site, 1, precursor mass tolerance, 8 ppm, fragment mass tolerance, 10 ppm, static modification, cysteine carbamidomethylation. The protein identification threshold was set to less than 1% for both peptide and protein false discovery rates. Peptide quantification was calculated with Scaffold DIA's EncyclopeDIA algorithm (PMID: 30510204). For each peptide, the four highest quality fragment ions were selected and quantified. Protein quantification was estimated from summed peptide quantification.
　本解析により、3875種類ものタンパク質の定量に成功した。この中から、以下の基準に従って、好酸球増多症又は気管支喘息バイオマーカーとなるタンパク質を抽出した。 Through this analysis, we succeeded in quantifying 3875 types of proteins. Proteins serving as biomarkers for eosinophilia or bronchial asthma were extracted from this according to the following criteria.
　気管支喘息被検体全体と健常被検体とを比較し、Welchのt検定により、2つの群間で有意に（P<0.05）且つ2倍以上離れた発現レベルを示すタンパク質を抽出した（表１及び表２）。 All subjects with bronchial asthma and healthy subjects were compared, and by Welch's t-test, proteins showing expression levels that were significantly (P<0.05) and more than two times apart between the two groups were extracted (Table 1 and Table 2).
　また、アトピー性喘息被検体と健常被検体とを比較し、Welchのt検定により、2つの群間で有意に（P<0.05）且つ2倍以上離れた発現レベルを示すタンパク質を抽出した（表３及び表４）。 In addition, atopic asthmatic subjects and healthy subjects were compared, and by Welch's t-test, proteins showing expression levels that were significantly (P<0.05) and more than two times apart between the two groups were extracted (Table 3 and Table 4).
　さらに、好酸球性喘息被検体と健常被検体とを比較し、Welchのt検定により、2つの群間で有意に（P<0.05）且つ2倍以上離れた発現レベルを示すタンパク質を抽出した（表５及び表６）。 Furthermore, eosinophilic asthma subjects and healthy subjects were compared, and by Welch's t-test, proteins showing significantly (P<0.05) and two-fold or more different expression levels between the two groups were extracted. (Tables 5 and 6).
　（結果）
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、気管支喘息被検体全体の発現レベルが健常被検体の発現レベルよりも高かったタンパク質を、表１（表１－１、表１－２、表１－３）に示す。表１中、「Fold change」は、気管支喘息被検体全体の発現量平均値を健常被検体の発現量平均値で除した値（気管支喘息被検体全体の発現量平均値／健常被検体の発現量平均値）を示す。 (result)
Among the extracted biomarkers for eosinophilia or bronchial asthma, proteins whose expression levels in all subjects with bronchial asthma were higher than those in healthy subjects are shown in Table 1 (Table 1-1, Table 1-2 , Table 1-3). In Table 1, "Fold change" is the value obtained by dividing the average expression level of all subjects with bronchial asthma by the average expression level of healthy subjects (average expression level of all subjects with bronchial asthma / expression level of healthy subjects). amount average value).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、気管支喘息被検体全体の発現レベルが健常被検体の発現レベルよりも低かったタンパク質を、表２に示す。表２中、「Fold change」は、健常被検体の発現量平均値を気管支喘息被検体全体の発現量平均値で除した値（健常被検体の発現量平均値／気管支喘息被検体全体の発現量平均値）を示す。 Among the extracted biomarkers for eosinophilia or bronchial asthma, Table 2 shows proteins whose expression levels in all bronchial asthma subjects were lower than in healthy subjects. In Table 2, "Fold change" is the value obtained by dividing the average expression level of healthy subjects by the average expression level of all subjects with bronchial asthma (average expression level of healthy subjects / expression level of all subjects with bronchial asthma). amount average value).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、アトピー性喘息被検体の発現レベルが健常被検体の発現レベルよりも高かったタンパク質を、表３（表３－１、表３－２）に示す。表３中、「Fold change」は、アトピー性喘息被検体の発現量平均値を健常被検体の発現量平均値で除した値（アトピー性喘息被検体の発現量平均値／健常被検体の発現量平均値）を示す。 Among the extracted biomarkers for eosinophilia or bronchial asthma, proteins whose expression levels in subjects with atopic asthma were higher than those in healthy subjects are shown in Table 3 (Tables 3-1 and 3-2). ). In Table 3, "Fold change" is the value obtained by dividing the average expression level of subjects with atopic asthma by the average expression level of healthy subjects (average expression level of subjects with atopic asthma/expression level of healthy subjects). amount average value).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、アトピー性喘息被検体の発現レベルが健常被検体の発現レベルよりも低かったタンパク質を、表４（表４－１、表４－２）に示す。表４中、「Fold change」は、健常被検体の発現量平均値をアトピー性喘息被検体の発現量平均値で除した値（健常被検体の発現量平均値／アトピー性喘息被検体の発現量平均値）を示す。 Among the extracted biomarkers for eosinophilia or bronchial asthma, proteins whose expression levels in subjects with atopic asthma were lower than those in healthy subjects are shown in Table 4 (Tables 4-1 and 4-2). ). In Table 4, "Fold change" is the value obtained by dividing the average expression level of healthy subjects by the average expression level of atopic asthma subjects (average expression level of healthy subjects / expression level of atopic asthma subjects amount average value).
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、好酸球性喘息被検体の発現レベルが健常被検体の発現レベルよりも高かったタンパク質を、表５に示す。表５中、「Fold change」は、好酸球性喘息被検体の発現量平均値を健常被検体の発現量平均値で除した値（好酸球性喘息被検体の発現量平均値／健常被検体の発現量平均値）を示す。 Table 5 shows proteins whose expression levels in subjects with eosinophilic asthma were higher than those in healthy subjects, among the extracted biomarkers for eosinophilia or bronchial asthma. In Table 5, "Fold change" is the value obtained by dividing the average expression level of subjects with eosinophilic asthma by the average expression level of healthy subjects (mean expression level of subjects with eosinophilic asthma/ The expression level average value of the subject) is shown.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　表５のバイオマーカーの中でも、Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonucleaseは、特に、アトピー性喘息との鑑別にも使用できるものであった（図４）。 Among the biomarkers in Table 5, Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, and Non-secretory ribonuclease could be used to differentiate from atopic asthma (Fig. 4). .
　抽出した好酸球増多症又は気管支喘息バイオマーカーの内、好酸球性喘息被検体の発現レベルが健常被検体の発現レベルよりも低かったタンパク質を、表６（表６－１、表６－２）に示す。表６中、「Fold change」は、健常被検体の発現量平均値を好酸球性喘息被検体の発現量平均値で除した値（健常被検体の発現量平均値／好酸球性喘息被検体の発現量平均値）を示す。 Among the extracted biomarkers for eosinophilia or bronchial asthma, proteins whose expression levels in subjects with eosinophilic asthma were lower than those in healthy subjects are shown in Table 6 (Table 6-1, Table 6 -2). In Table 6, "Fold change" is the value obtained by dividing the average expression level of healthy subjects by the average expression level of eosinophilic asthma subjects (average expression level of healthy subjects/eosinophilic asthma The expression level average value of the subject) is shown.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　試験例3．好酸球増多症又は気管支喘息バイオマーカーの評価1
　試験例1の各検体における、Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinそれぞれの好酸球増多症又は気管支喘息バイオマーカーの発現量と血中好酸球数に基づいて、好酸球増多症又は気管支喘息バイオマーカーの発現量と好酸球数との相関係数を算出した。 Test example 3. Evaluation of eosinophilia or bronchial asthma biomarkers1
Galectin-10, bone marrow proteoglycan, eosinophil peroxidase, non-secretory ribonuclease, and eosinophil cationic protein expression levels of eosinophilia or bronchial asthma biomarkers and blood eosinophils in each sample of Test Example 1 Based on the numbers, correlation coefficients between the expression levels of eosinophilia or bronchial asthma biomarkers and the number of eosinophils were calculated.
　その結果、Galectin-10の発現量と好酸球数との相関係数は0.95であり、Bone marrow proteoglycanの発現量と好酸球数との相関係数は0.93であり、Eosinophil peroxidaseの発現量と好酸球数との相関係数は0.88であり、Non-secretory ribonucleaseの発現量と好酸球数との相関係数は0.88であり、Eosinophil cationic proteinの発現量と好酸球数との相関係数は0.44であった。 As a result, the correlation coefficient between the expression level of Galectin-10 and the eosinophil count was 0.95, the correlation coefficient between the expression level of bone marrow proteoglycan and the eosinophil count was 0.93, and the expression level of eosinophil peroxidase was 0.93. and the eosinophil count was 0.88, the correlation coefficient between the expression level of non-secretory ribonuclease and the eosinophil count was 0.88, and the correlation coefficient between the expression level of eosinophil catic protein and the eosinophil count was 0.88. The correlation coefficient was 0.44.
　試験例4．好酸球増多症又は気管支喘息バイオマーカーの評価2
　試験例2の定量結果に基づいて、Galectin-10を単独で気管支喘息バイオマーカーとして使用した際のROC曲線を作製した。また、ポジティブコントロールとして、同じ被検体についての血中好酸球数を気管支喘息バイオマーカーとして使用した際のROC曲線を作製した。具体的には、縦軸を感度（陽性率）とし、横軸を1から特異度を減じた値（1－特異度）（偽陽性率）とするROC曲線を統計ソフトJMPを用いて作成した。 Test example 4. Evaluation of eosinophilia or bronchial asthma biomarkers2
Based on the quantitative results of Test Example 2, an ROC curve was prepared when Galectin-10 was used alone as a biomarker for bronchial asthma. Also, as a positive control, an ROC curve was prepared for the same subject using the blood eosinophil count as a bronchial asthma biomarker. Specifically, an ROC curve with sensitivity (positive rate) on the vertical axis and a value obtained by subtracting specificity from 1 (1 - specificity) (false positive rate) on the horizontal axis was created using the statistical software JMP. .
　その結果、Galectin-10をバイオマーカーとして使用した場合のAUCは0.96875であり、血中好酸球数をバイオマーカーとして使用した場合のAUCは0.93750であった。このことから、Galectin-10は好酸球数に比べて喘息の診断能が高いことが分かった。 　As a result, the AUC was 0.96875 when Galectin-10 was used as a biomarker, and the AUC was 0.93750 when the blood eosinophil count was used as a biomarker. From this, it was found that galectin-10 has a higher diagnostic ability for asthma than the eosinophil count.
　試験例5．好酸球増多症又は気管支喘息バイオマーカーの評価3
　試験例1の各検体における、Galectin-10等の好酸球増多症又は気管支喘息バイオマーカーの発現量及び血中好酸球数と、呼吸機能（FEV1％）との相関係数を算出した。 Test example 5. Evaluation of eosinophilia or bronchial asthma biomarkers3
The correlation coefficient between the expression level of biomarkers for eosinophilia or bronchial asthma such as Galectin-10, blood eosinophil count, and respiratory function (FEV1%) was calculated for each sample in Test Example 1. .
　その結果、Galectin-10の発現量とFEV1％との相関係数は－0.8344（p値0.0014）であり、好酸球数とFEV1％との相関係数は－0.8073（p値0.0027）であった。また、他の好酸球増多症又は気管支喘息バイオマーカーについても、FEV1％との相関が認められた。 As a result, the correlation coefficient between the expression level of Galectin-10 and FEV1% was -0.8344 (p-value 0.0014), and the correlation coefficient between eosinophil count and FEV1% was -0.8073 (p-value 0.0027). Ta. Other eosinophilia or bronchial asthma biomarkers were also correlated with FEV1%.
　試験例6．好酸球増多症又は気管支喘息バイオマーカーの評価4
　難治性喘息には、好酸球性副鼻腔炎が合併することがある。好酸球性副鼻腔炎は、予後が悪い、鼻茸を伴う慢性副鼻腔炎（chronic rhinosinusitis with nasal polyp: CRSwNP）と、予後が良好である、鼻茸を伴わない慢性副鼻腔炎（chronic rhinosinusitis without nasal polyp: CRSsNP）とに分類される。そこで、好酸球増多症又は気管支喘息バイオマーカーによりこれらの鑑別が可能か調べた。 Test example 6. Evaluation of eosinophilia or bronchial asthma biomarkers4
Refractory asthma may be complicated by eosinophilic sinusitis. Eosinophilic sinusitis is classified into chronic rhinosinusitis with nasal polyp (CRSwNP), which has a poor prognosis, and chronic rhinosinusitis without nasal polyp (CRSwNP), which has a favorable prognosis. polyp: CRSsNP). Therefore, we investigated whether it is possible to distinguish between these by eosinophilia or bronchial asthma biomarkers.
　CRSwNPヒト被検体7名とCRSsNPヒト被検体6名を準備した。被検体のプロファイルを表７に示す。 We prepared 7 CRSwNP human subjects and 6 CRSsNP human subjects. Subject profiles are shown in Table 7.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　CRSwNPヒト被検体7名とCRSsNPヒト被検体6名から試験例1と同様にして細胞外小胞画分（Exosome）及び組織（下鼻甲介及びpolyp）を採取し、これらのサンプル中のGalectin-10等の好酸球増多症又は気管支喘息バイオマーカーの発現量を測定した。結果を図１及び図２に示す。CRSwNPのエクソソーム及び組織療法においてGalectin-10が増加していることが分かった。 Extracellular vesicle fractions (Exosomes) and tissues (inferior turbinate and polyp) were collected from 7 CRSwNP human subjects and 6 CRSsNP human subjects in the same manner as in Test Example 1, and Galectin- Expression levels of eosinophilia or bronchial asthma biomarkers such as 10 were measured. The results are shown in FIGS. 1 and 2. FIG. Galectin-10 was found to be increased in exosome and tissue therapy of CRSwNP.
　上記細胞外小胞画分及び組織について、試験例2と同様にしてプロテオミクス解析を行った。その結果、細胞外小胞画分及び組織のいずれでも、CRSwNPにおいて有意に発現レベルが高かったタンパク質が11個見出された（表８）。 Proteomics analysis was performed in the same manner as in Test Example 2 for the above extracellular vesicle fractions and tissues. As a result, 11 proteins whose expression levels were significantly high in CRSwNP were found in both extracellular vesicle fractions and tissues (Table 8).
Figure JPOXMLDOC01-appb-T000013
 　上記11個のタンパク質の中でも、5つのタンパク質については、細胞外小胞画分と組織間で発現量に相関（r＞0.5）があった（図１１）。
Figure JPOXMLDOC01-appb-T000013
 Among the 11 proteins mentioned above, there was a correlation (r>0.5) in the expression levels of 5 proteins between the extracellular vesicle fraction and the tissue (Fig. 11).
　次に、血中好酸球数と組織中のGalectin-10発現量との相関係数、及びエクソソーム中のGalectin-10発現量と組織中のGalectin-10発現量との相関係数を算出した。その結果、血中好酸球数と組織中のGalectin-10発現量との相関係数は0.5409（p値0.0858）であり、エクソソーム中のGalectin-10発現量と組織中のGalectin-10発現量との相関係数は0.6709（p値0.0337）であった。このことから、エクソソーム中のGalectin-10発現量は、血中好酸球数よりも、組織中のGalectin-10発現量に強く相関していることが分かった。 Next, the correlation coefficient between the number of blood eosinophils and the Galectin-10 expression level in tissues, and the correlation coefficient between the Galectin-10 expression level in exosomes and the Galectin-10 expression level in tissues were calculated. . As a result, the correlation coefficient between blood eosinophil count and Galectin-10 expression level in tissues was 0.5409 (p-value 0.0858), indicating that Galectin-10 expression level in exosomes and Galectin-10 expression level in tissues was 0.6709 (p-value 0.0337). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with the Galectin-10 expression level in tissues rather than the blood eosinophil count.
　続いて、血中好酸球数と組織好酸球浸潤（/HPF）との相関係数、及びエクソソーム中のGalectin-10発現量と組織好酸球浸潤（/HPF）との相関係数を算出した。その結果、血中好酸球数と組織好酸球浸潤（/HPF）との相関係数は0.6053（p値0.0284）であり、エクソソーム中のGalectin-10発現量と組織好酸球浸潤（/HPF）との相関係数は0.7534（p値0.0119）であった。このことから、エクソソーム中のGalectin-10発現量は、血中好酸球数よりも、組織好酸球浸潤に強く相関していることが分かった。 Subsequently, the correlation coefficient between blood eosinophil count and tissue eosinophil infiltration (/HPF), and the correlation coefficient between Galectin-10 expression level in exosomes and tissue eosinophil infiltration (/HPF) Calculated. As a result, the correlation coefficient between the number of blood eosinophils and tissue eosinophil infiltration (/HPF) was 0.6053 (p-value 0.0284), and the expression level of Galectin-10 in exosomes and tissue eosinophil infiltration (/ HPF) was 0.7534 (p-value 0.0119). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with tissue eosinophil infiltration rather than the blood eosinophil count.
　続いて、血中好酸球数とJESREC scoreとの相関係数、及びエクソソーム中のGalectin-10発現量とJESREC scoreとの相関係数を算出した。その結果、血中好酸球数とJESREC scoreとの相関係数は0.7622（p値0.0025）であり、エクソソーム中のGalectin-10発現量とJESREC scoreとの相関係数は0.7791（p値0.0079）であった。 Next, we calculated the correlation coefficient between blood eosinophil count and JESREC score, and the correlation coefficient between Galectin-10 expression level in exosomes and JESREC score. As a result, the correlation coefficient between blood eosinophil count and JESREC score was 0.7622 (p-value 0.0025), and the correlation coefficient between Galectin-10 expression level in exosomes and JESREC score was 0.7791 (p-value 0.0079). Met.
　試験例7．好酸球増多症又は気管支喘息バイオマーカーの評価5
　マウス喘息モデルにおいて、EETosisの抑制が喘息の病態悪化を抑制することが報告されている（Nature Cell Biology volume 23, pages 1060-1072 (2021)）。そこで、試験例6の被検体について、上記文献に記載の方法に従って組織中のEETosis陽性好酸球数（/HPF）を測定し、血中好酸球数及びエクソソーム中のGalectin-10発現量との相関係数を算出した。その結果、血中好酸球数と組織中のEETosis陽性好酸球数（/HPF）との相関係数は0.5522（p値0.0504）であり、エクソソーム中のGalectin-10発現量と組織中のEETosis陽性好酸球数（/HPF）との相関係数は0.7886（p値0.0067）であった。このことから、エクソソーム中のGalectin-10発現量は、血中好酸球数よりも、EETosis陽性好酸球数に強く相関していることが分かった。 Test example 7. Evaluation of eosinophilia or bronchial asthma biomarkers5
It has been reported that suppression of EETosis suppresses exacerbation of asthma in a mouse asthma model (Nature Cell Biology volume 23, pages 1060-1072 (2021)). Therefore, for the subject of Test Example 6, the number of EETosis-positive eosinophils (/HPF) in the tissue was measured according to the method described in the above document, and the number of blood eosinophils and the expression level of Galectin-10 in exosomes The correlation coefficient of was calculated. As a result, the correlation coefficient between the number of blood eosinophils and the number of EETosis-positive eosinophils in tissues (/HPF) was 0.5522 (p value 0.0504). The correlation coefficient with EETosis-positive eosinophil count (/HPF) was 0.7886 (p-value 0.0067). From this, it was found that the Galectin-10 expression level in exosomes correlated more strongly with the number of EETosis-positive eosinophils than the number of blood eosinophils.
　試験例8．好酸球増多症又は気管支喘息バイオマーカーの評価6
　in vitroにおける好酸球活性化によりエクソソーム中のGalectin-10が増加するかどうかを調べた。具体的には、既報（Nat Protoc . 2006;1(6):2613-20.、Blood . 2013 Mar 14;121(11):2074-83.）に従って、好酸球を単離し、EETosisを誘導した後、上清由来エクソソームのウェスタンブロッティングを行った。 Test example 8. Evaluation of eosinophilia or bronchial asthma biomarkers6
We investigated whether in vitro eosinophil activation increases Galectin-10 in exosomes. Specifically, according to previous reports (Nat Protoc. 2006; 1(6): 2613-20., Blood. 2013 Mar 14; 121(11): 2074-83.), eosinophils were isolated and EETosis was induced. After that, Western blotting of supernatant-derived exosomes was performed.
　結果を図３に示す。in vitroでも好酸球の状態をエクソソームが反映していることが分かった。 The results are shown in Figure 3. Exosomes were also found to reflect the state of eosinophils in vitro.
　試験例9．好酸球増多症又は気管支喘息バイオマーカーの評価7
　健常被検体（n＝9）、COPD被検体（n＝7）、及び喘息被検体（n＝50）それぞれの血清から精製したエクソソーム（試験例1と同様にして精製）を用いて、Galectin-10抗体によるイムノブロッティングを行った。 Test example 9. Evaluation of eosinophilia or bronchial asthma biomarkers7
Using exosomes purified from serum of healthy subjects (n = 9), COPD subjects (n = 7), and asthma subjects (n = 50) (purified in the same manner as in Test Example 1), Galectin- Immunoblotting with 10 antibodies was performed.
　結果を図５に示す。Galectin-10はイムノブロッティングによっても検出可能であり、喘息と、健常、COPDとを判定できることが分かった。 The results are shown in Figure 5. Galectin-10 was also detectable by immunoblotting, and it was found that asthma, normal, and COPD could be determined.
　試験例10．好酸球増多症又は気管支喘息バイオマーカーの評価8
　健常被検体（n＝5）、及び喘息被検体（n＝6）それぞれの肺組織を用いて、Galectin-10抗体による蛍光免疫染色を行い、Galectin-10抗体で染色される領域の面積を測定した。 Test example 10. Evaluation of eosinophilia or bronchial asthma biomarkers8
Lung tissue from healthy subjects (n = 5) and asthmatic subjects (n = 6) was immunofluorescently stained with Galectin-10 antibody, and the area of the region stained with Galectin-10 antibody was measured. did.
　結果を図６に示す。喘息肺組織内においてGalectin-10が増加することが分かった。 The results are shown in Figure 6. Galectin-10 was found to increase in asthmatic lung tissue.
　試験例11．好酸球増多症又は気管支喘息バイオマーカーの評価9
　LCMD（Laser capture microdissection）で手術肺（健常被検体（n＝3）、及び喘息被検体（n＝4））の気管支周囲を切離し、プロテオミクス（LC-MS/MS）でGalectin-10を定量した。 Test example 11. Evaluation of eosinophilia or bronchial asthma biomarkers9
Galectin-10 was quantified by proteomics (LC-MS/MS) after peribronchial dissection of operated lungs (healthy subjects (n = 3) and asthmatic subjects (n = 4)) by LCMD (Laser capture microdissection). .
　結果を図７に示す。喘息気管支周囲においてGalectin-10が増加することが分かった。 The results are shown in Figure 7. Galectin-10 was found to increase in peribronchial asthma.
　試験例12．好酸球増多症又は気管支喘息バイオマーカーの評価10
　好酸球浸潤は、粘液腺の形成を引き起こし、これにより気道壁肥厚が惹起され、閉塞性変化に至ることが分かっている。そこで、これらの現象とGalectin-10との相関を調べた。具体的には、喘息患者（n＝55）について、血清エクソソーム中のGalectin-10の発現レベル及び血中好酸球数を測定し、さらに気管支壁肥厚の程度（WA％：図８に測定・算出方法示す）と呼吸機能（FEV1％）を測定し、相関係数rを算出した。
　結果を図９及び図１０に示す。Galectin-10は、好酸球数よりも、気道壁肥厚及び閉塞性変化と相関していることが分かった。 Test example 12. Evaluation of eosinophilia or bronchial asthma biomarkers10
Eosinophil infiltration has been shown to cause the formation of mucous glands, which induce airway wall thickening and lead to obstructive changes. Therefore, we investigated the correlation between these phenomena and Galectin-10. Specifically, for asthmatic patients (n = 55), the expression level of Galectin-10 in serum exosomes and the number of blood eosinophils were measured, and the degree of bronchial wall thickening (WA%: Calculation method shown) and respiratory function (FEV1%) were measured, and the correlation coefficient r was calculated.
The results are shown in FIGS. 9 and 10. FIG. Galectin-10 was found to be more correlated with airway wall thickening and obstructive changes than with eosinophil counts.

Claims (17)

  1. 好酸球増多症又は気管支喘息を検査する方法であって、
    （1）被検体から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
    （A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
    （B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
    （C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
    （D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
    （E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
    （F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
    （G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質を検出する工程を含む、検査方法。     A method of testing for eosinophilia or bronchial asthma, comprising:
    (1) Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D), protein in extracellular vesicles of body fluid collected from a subject or body fluid Group (E), Protein Group (F), and Protein Group (G):
    (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
    (B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
    (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
    (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
    (E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
    (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
    A testing method comprising the step of detecting at least one protein selected from the group consisting of
  2. 検査対象疾患が呼吸器疾患である、請求項１に記載の検査方法。 The examination method according to claim 1, wherein the disease to be examined is a respiratory disease.
  3. 検査対象疾患が気管支喘息である、請求項１又は２に記載の検査方法。 3. The test method according to claim 1 or 2, wherein the disease to be tested is bronchial asthma.
  4. 検査対象疾患が好酸球性喘息である、請求項１～３のいずれかに記載の検査方法。 The test method according to any one of claims 1 to 3, wherein the disease to be tested is eosinophilic asthma.
  5. 検出するタンパク質が、タンパク質群（F）及びタンパク質群（G）からなる群より選択される少なくとも1種のタンパク質を含む、請求項４に記載の検査方法。 The inspection method according to claim 4, wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (F) and protein group (G).
  6. 検出したタンパク質が、タンパク質群（F）より選択される少なくとも1種のタンパク質（タンパク質F’）を含む場合、さらに、
    （2F）前記工程（1）で検出されたタンパク質F’の量又は濃度がカットオフ値以上である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
    を含む、請求項４又は５に記載の検査方法。     When the detected protein contains at least one protein (protein F') selected from the protein group (F),
    (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value;
    The inspection method according to claim 4 or 5, comprising:
  7. 検出したタンパク質が、タンパク質群（G）より選択される少なくとも1種のタンパク質（タンパク質G’）を含む場合、さらに、
    （2G）前記工程（1）で検出されたタンパク質G’の量又は濃度がカットオフ値以下である場合に、前記被検体が好酸球性喘息に罹患していると判定する工程、
    を含む、請求項４～６のいずれかに記載の検査方法。     When the detected protein contains at least one protein (protein G') selected from the protein group (G),
    (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value;
    The inspection method according to any one of claims 4 to 6, comprising.
  8. 検査対象疾患がアトピー性喘息である、請求項１～３のいずれかに記載の検査方法。 The examination method according to any one of claims 1 to 3, wherein the disease to be examined is atopic asthma.
  9. 検出するタンパク質が、タンパク質群（D）及びタンパク質群（E）からなる群より選択される少なくとも1種のタンパク質を含む、請求項８に記載の検査方法。 The inspection method according to claim 8, wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (D) and protein group (E).
  10. 検出したタンパク質が、タンパク質群（D）より選択される少なくとも1種のタンパク質（タンパク質D’）を含む場合、さらに、
    （2D）前記工程（1）で検出されたタンパク質D’の量又は濃度がカットオフ値以上である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
    を含む、請求項８又は９に記載の検査方法。     When the detected protein contains at least one protein (protein D') selected from the protein group (D),
    (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value;
    The inspection method according to claim 8 or 9, comprising:
  11. 検出したタンパク質が、タンパク質群（E）より選択される少なくとも1種のタンパク質（タンパク質E’）を含む場合、さらに、
    （2E）前記工程（1）で検出されたタンパク質E’の量又は濃度がカットオフ値以下である場合に、前記被検体がアトピー性喘息に罹患していると判定する工程、
    を含む、請求項８～１０のいずれかに記載の検査方法。     When the detected protein contains at least one protein (protein E') selected from protein group (E),
    (2E) determining that the subject is suffering from atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value;
    The inspection method according to any one of claims 8 to 10, comprising.
  12. 検出するタンパク質が、タンパク質群（A）を含む、請求項１～１１のいずれかに記載の検査方法。 The test method according to any one of claims 1 to 11, wherein the protein to be detected comprises protein group (A).
  13. 検出したタンパク質が、タンパク質群（A）より選択される少なくとも1種のタンパク質（タンパク質A’）を含む場合、さらに、
    （2A）前記工程（1）で検出されたタンパク質A’の量又は濃度がカットオフ値以上である場合に、前記被検体が高重症度の好酸球増多症又は気管支喘息に罹患している又は高重症度の好酸球増多症又は気管支喘息に将来罹患する可能性が高いと判定する工程、
    を含む、請求項１～１２のいずれかに記載の検査方法。     When the detected protein contains at least one protein (protein A') selected from protein group (A),
    (2A) when the amount or concentration of protein A' detected in step (1) is equal to or higher than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future;
    The inspection method according to any one of claims 1 to 12, comprising.
  14. タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
    （A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
    （B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
    （C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
    （D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
    （E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
    （F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
    （G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、好酸球増多症又は気管支喘息の検査薬。     Protein Group (A), Protein Group (X), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), and Protein Group (G):
    (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
    (B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
    (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
    (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
    (E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
    (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
    A test agent for eosinophilia or bronchial asthma, comprising at least one protein-detecting agent selected from the group consisting of:
  15. タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（D）、並びにタンパク質群（F）：
    （A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
    （B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
    （D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、並びに
    （F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質の抑制剤、並びに
    タンパク質群（C）、タンパク質群（E）、並びにタンパク質群（G）：
    （C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
    （E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、並びに
    （G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質の亢進剤
    からなる群より選択される少なくとも1種の薬剤を含有する、好酸球増多症又は気管支喘息の予防又は治療剤。     Protein Group (A), Protein Group (X), Protein Group (B), Protein Group (D), and Protein Group (F):
    (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
    (B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
    (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- Protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2, and (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease , Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig -like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum-Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A protein group,
    An inhibitor of at least one protein selected from the group consisting of, as well as protein group (C), protein group (E), and protein group (G):
    (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
    (E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, Protein group consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, Synaptotagmin-like protein 4, and charged multivesicular body protein 2a, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ- associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein , STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 A protein group consisting of member 4, Solute carrier family 12 member 7, and Solute carrier family 43 member 3,
    A prophylactic or therapeutic agent for eosinophilia or bronchial asthma, containing at least one drug selected from the group consisting of at least one protein enhancer selected from the group consisting of:
  16. 被検物質で処理された動物から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（X）、タンパク質群（B）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
    （A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
    （B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
    （C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
    （D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
    （E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
    （F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
    （G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の予防又は治療剤の有効成分のスクリーニング方法。     Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D ), Protein Group (E), Protein Group (F), and Protein Group (G):
    (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
    (B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
    (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
    (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
    (E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
    (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
    A method of screening for an active ingredient of a prophylactic or therapeutic agent for eosinophilia or bronchial asthma, wherein the amount or concentration of at least one protein selected from the group consisting of is used as an indicator.
  17. 被検物質で処理された動物から採取された体液の細胞外小胞又は体液における、タンパク質群（A）、タンパク質群（B）、タンパク質群（X）、タンパク質群（C）、タンパク質群（D）、タンパク質群（E）、タンパク質群（F）、並びにタンパク質群（G）：
    （A）Galectin-10、Bone marrow proteoglycan、Eosinophil peroxidase、Non-secretory ribonuclease、及びEosinophil cationic proteinからなるタンパク質群、(X)Cholinesterase、Galectin-10、Exportin-2、Eosinophil peroxidase、Coagulation factor XII、Ficolin-3、Immunoglobulin heavy variable 4-28、Immunoglobulin heavy variable 6-1、Immunoglobulin heavy variable 7-43、Immunoglobulin heavy variable 8-61、及びImmunoglobulin heavy variable 9-49からなるタンパク質群、
    （B）HLA class II histocompatibility antigen, DRB1-9 beta chain、Galectin-10、Eosinophil peroxidase、Protein mono-ADP-ribosyltransferase PARP4、Non-secretory ribonuclease、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、Immunoglobulin heavy constant gamma 2、Cytochrome P450 4F8、C-type lectin domain family 12 member A、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-9、Immunoglobulin lambda variable 3-21、Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Prelamin-A/C、CMRF35-like molecule 1、Eosinophil cationic protein、GDH/6PGL endoplasmic bifunctional protein、Low affinity immunoglobulin gamma Fc region receptor III-A、Urokinase plasminogen activator surface receptor、Voltage-gated hydrogen channel 1、RNA polymerase-associated protein RTF1 homolog、Cytochrome P450 4F3、Neutrophil cytosol factor 4、Activated RNA polymerase II transcriptional coactivator p15、Cell surface glycoprotein CD200 receptor 1、Major vault protein、C-type lectin domain family 5 member A、Disintegrin and metalloproteinase domain-containing protein 8、Zinc finger Ran-binding domain-containing protein 2、Myeloid cell surface antigen CD33、Heme oxygenase 2、Lysosomal acid glucosylceramidase、Cartilage intermediate layer protein 1、Sialic acid-binding Ig-like lectin 5、Arachidonate 5-lipoxygenase、Estradiol 17-beta-dehydrogenase 11、Torsin-1A、Cytochrome b-245 light chain、Lysosomal-trafficking regulator、Liver carboxylesterase 1、Resistin、Immunoglobulin heavy constant alpha 1、Torsin-1A-interacting protein 1、Hexokinase-3、Cytidine deaminase、Matrix metalloproteinase-25、Aldehyde dehydrogenase family 3 member B1、Platelet basic protein、Type-1 angiotensin II receptor-associated protein、Metalloreductase STEAP4、Protein S100-P、Cytochrome b-245 heavy chain、Immunoglobulin alpha Fc receptor、Phospholipase B-like 1、Lysophosphatidylcholine acyltransferase 2、Arachidonate 5-lipoxygenase-activating protein、Coagulation factor XII、Lysophospholipid acyltransferase 7、Protein S100-A9、Protein DEK、Neutrophil defensin 1、Neutrophil defensin 1、Neutrophil defensin 3、Serum amyloid A-4 protein、Erlin-2、Leukotriene B4 receptor 1、Epoxide hydrolase 1、Microsomal glutathione S-transferase 1、Tyrosine-protein phosphatase non-receptor type substrate 1、Complement factor D、PITH domain-containing protein 1、Annexin A1、Piezo-type mechanosensitive ion channel component 1、Apolipoprotein B receptor、Protein S100-A11、Apoptosis-associated speck-like protein containing a CARD、Long-chain-fatty-acid--CoA ligase 1、Glycerol-3-phosphate acyltransferase 3、Alpha-1-acid glycoprotein 1、Anoctamin-10、Leucine-rich repeat and calponin homology domain-containing protein 4、及びVery-long-chain 3-oxoacyl-CoA reductaseからなるタンパク質群、
    （C）Thioredoxin domain-containing protein 12、Bone morphogenetic protein 10、Butyrophilin subfamily 3 member A2、Mitogen-activated protein kinase kinase kinase 7、Methyltransferase-like 26、von Willebrand factor A domain-containing protein 5A、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Gamma-enolase、LHFPL tetraspan subfamily member 6 protein、FYVE, RhoGEF and PH domain-containing protein 4、Sorcin、Tetraspanin-33、Tight junction protein ZO-2、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Disintegrin and metalloproteinase domain-containing protein 10、Choline transporter-like protein 2、Probable UDP-sugar transporter protein SLC35A5、WD repeat-containing protein 11、及びCanalicular multispecific organic anion transporter 2からなるタンパク質群、
    （D）Putative inactive carboxylesterase 4、Neutrophil cytosol factor 2、Immunoglobulin heavy constant gamma 2、Prelamin-A/C、C-type lectin domain family 12 member A、Low affinity immunoglobulin gamma Fc region receptor III-A、Non-secretory ribonuclease、Cartilage intermediate layer protein 1、Galectin-10、Resistin、Estradiol 17-beta-dehydrogenase 11、Bactericidal permeability-increasing protein、Cell surface glycoprotein CD200 receptor 1、Putative neutrophil cytosol factor 1B、Putative neutrophil cytosol factor 1B、Neutrophil cytosol factor 1、Neutrophil cytosol factor 4、Matrix metalloproteinase-25、Heme oxygenase 2、Urokinase plasminogen activator surface receptor、Epoxide hydrolase 1、Torsin-1A-interacting protein 1、Arachidonate 5-lipoxygenase、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Amyloid-beta precursor protein、Arachidonate 5-lipoxygenase-activating protein、Alpha-1-acid glycoprotein 1、Protein S100-A9、Perilipin-1、Protein DEK、Protein S100-P、ELMO domain-containing protein 2、Microsomal glutathione S-transferase 1、Phospholipase B-like 1、Cytochrome b-245 heavy chain、Merlin、Complement factor I、Cytochrome b-245 light chain、Casein kinase I isoform gamma-1、Inter-alpha-trypsin inhibitor heavy chain H1、Long-chain-fatty-acid--CoA ligase 1、及びInter-alpha-trypsin inhibitor heavy chain H2からなるタンパク質群、
    （E）HLA class II histocompatibility antigen, DRB1-4 beta chain、Gasdermin-D、MAP/microtubule affinity-regulating kinase 3、Thioredoxin domain-containing protein 12、Methyltransferase-like 26、LIM domain and actin-binding protein 1、Serine/threonine-protein phosphatase PP1-gamma catalytic subunit、Methionine--tRNA ligase, cytoplasmic、Butyrophilin subfamily 3 member A2、FYVE, RhoGEF and PH domain-containing protein 4、Katanin p60 ATPase-containing subunit A-like 1、von Willebrand factor A domain-containing protein 5A、Tight junction protein ZO-2、Proteinase-activated receptor 1、Gamma-enolase、Mitogen-activated protein kinase kinase kinase 7、Cytohesin-2、Immunoglobulin-binding protein 1、Armadillo repeat-containing protein 8、Nucleolysin TIA-1 isoform p40、Nucleolysin TIA-1 isoform p40、Nucleolysin TIAR、Integrin beta-3、GMP reductase 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、Coactosin-like protein、Integrin alpha-IIb、Protein cereblon、PDZ and LIM domain protein 1、Basic leucine zipper and W2 domain-containing protein 1、LHFPL tetraspan subfamily member 6 protein、WD repeat-containing protein 11、Tyrosine-protein kinase BTK、DENN domain-containing protein 4C、N-acetyl-D-glucosamine kinase、Cofilin-1、Translin-associated protein X、Choline transporter-like protein 2、Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11、ADP-ribosylation factor 4、FYN-binding protein 1、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D3、Ubiquitin-conjugating enzyme E2 D2、Caveolae-associated protein 2、EH domain-containing protein 3、EH domain-containing protein 1、cAMP-dependent protein kinase type I-alpha regulatory subunit、Synaptotagmin-like protein 4、及びCharged multivesicular body protein 2aからなるタンパク質群、
    （F）Galectin-10、Eosinophil peroxidase、Bone marrow proteoglycan、Arachidonate 15-lipoxygenase、Non-secretory ribonuclease、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2D-29、Immunoglobulin kappa variable 2-29、2'-deoxynucleoside 5'-phosphate N-hydrolase 1、Voltage-gated hydrogen channel 1、Eosinophil cationic protein、Cytochrome b-245 light chain、Disintegrin and metalloproteinase domain-containing protein 8、Aldehyde dehydrogenase family 3 member B1、Cytidine deaminase、Heme oxygenase 2、Immunoglobulin heavy constant alpha 1、Metalloreductase STEAP4、Type-1 angiotensin II receptor-associated protein、Sialic acid-binding Ig-like lectin 5、Torsin-1A-interacting protein 1、Annexin A1、Estradiol 17-beta-dehydrogenase 11、Endoplasmic reticulum-Golgi intermediate compartment protein 1、Erlin-2、Anoctamin-10、及びRas-related protein Rab-27Aからなるタンパク質群、並びに
    （G）Bone morphogenetic protein 10、Thioredoxin domain-containing protein 12、Intermediate conductance calcium-activated potassium channel protein 4、Probable UDP-sugar transporter protein SLC35A5、Golgi integral membrane protein 4、Protein CASC4、Putative tyrosine-protein phosphatase auxilin、Butyrophilin subfamily 3 member A2、C1GALT1-specific chaperone 1、Protein FAM234B、Tetraspanin-33、Canalicular multispecific organic anion transporter 2、Mitogen-activated protein kinase kinase kinase 7、Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase、Wiskott-Aldrich syndrome protein family member 1、cGMP-inhibited 3',5'-cyclic phosphodiesterase A、DAZ-associated protein 1、C-type lectin domain family 2 member L、Leucyl-cystinyl aminopeptidase、Progressive ankylosis protein homolog、Disintegrin and metalloproteinase domain-containing protein 10、Calpain-7、Choline transporter-like protein 1、LHFPL tetraspan subfamily member 6 protein、STARD3 N-terminal-like protein、Inactive rhomboid protein 2、Serine incorporator 3、Annexin A4、Cas scaffolding protein family member 4、Low-density lipoprotein receptor-related protein 10、Tetraspanin-32、von Willebrand factor A domain-containing protein 5A、Gamma-enolase、MICAL-like protein 2、Vacuolar protein sorting-associated protein 4B、Metalloreductase STEAP3、Choline transporter-like protein 2、Protein transport protein Sec24C、Bis(5'-adenosyl)-triphosphatase ENPP4、Solute carrier family 12 member 4、Solute carrier family 12 member 7、及びSolute carrier family 43 member 3からなるタンパク質群、
    からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、好酸球増多症又は気管支喘息の誘発性又は増悪性の評価方法。     Protein group (A), protein group (B), protein group (X), protein group (C), protein group (D ), Protein Group (E), Protein Group (F), and Protein Group (G):
    (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49,
    (B) HLA class II histocompatibility antigen, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 member A, Immunoglobulin lambda variable 3- 9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Prelamin-A/C, CMRF35-like molecule 1, Eosinophil cationic protein, GDH/6PGL endoplasmic bifunctional protein, Low affinity immunoglobulin gamma Fc region receptor III-A, Urokinase plasminogen activator surface receptor, Voltage-gated hydrogen channel 1, RNA polymerase-associated protein RTF1 homolog, Cytochrome P450 4F3, Neutrophil cytosol factor 4, Activated RNA polymerase II transcriptional coactivator p15, Cell surface glycoprotein CD200 receptor 1, Major vault protein, C-type lectin domain family 5 member A, Disintegrin and metalloproteinase domain-containing protein 8, Zinc finger Ran-binding domain- containing protein 2, Myeloid cell surface antigen CD33, Heme oxygenase 2, Lysosomal acid glucosylceramidase, Cartilage intermediate layer protein 1, Sialic acid-binding Ig-like lectin 5, Arachidonate 5-lipoxygenase, Estradiol 17-beta-dehydrogenase 11, Torsin-1A , Cytochrome b-245 light chain, Lysosomal-trafficking regulator, Liver carboxylesterase 1, Resistin, Immunoglobulin heavy constant alpha 1, Torsin-1A-interacting protein 1, Hexokinase-3, Cytidine deaminase, Matrix metalloproteinase-25, Aldehyde dehydrogenase family 3 members B1, Platelet basic protein, Type-1 angiotensin II receptor-associated protein, Metalloreductase STEAP4, Protein S100-P, Cytochrome b-245 heavy chain, Immunoglobulin alpha Fc receptor, Phospholipase B-like 1, Lysophosphatidylcholine acyltransferase 2, Arachidonate 5-lipoxygenase -activating protein, Coagulation factor XII, Lysophospholipid acyltransferase 7, Protein S100-A9, Protein DEK, Neutrophil defensin 1, Neutrophil defensin 1, Neutrophil defensin 3, Serum amyloid A-4 protein, Erlin-2, Leukotriene B4 receptor 1, Epoxide hydrolase 1, Microsomal glutathione S-transferase 1, Tyrosine-protein phosphatase non-receptor type substrate 1, Complement factor D, PITH domain-containing protein 1, Annexin A1, Piezo-type mechanosensitive ion channel component 1, Apolipoprotein B receptor, Protein S100- A11, Apoptosis-associated speck-like protein containing a CARD, Long-chain-fatty-acid--CoA ligase 1, Glycerol-3-phosphate acyltransferase 3, Alpha-1-acid glycoprotein 1, Anoctamin-10, Leucine-rich repeat and calponin homology domain-containing protein 4, and a group of proteins consisting of Very-long-chain 3-oxoacyl-CoA reductase,
    (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, Nucleolysin TIA-1 isoform p40, Consists of Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Disintegrin and metalloproteinase domain-containing protein 10, Choline transporter-like protein 2, Probable UDP-sugar transporter protein SLC35A5, WD repeat-containing protein 11, and Canalicular multispecific organic anion transporter 2 protein group,
    (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III-A, Non-secretory ribonuclease , Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1 , Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 , Amyloid-beta precursor protein, Arachidonate 5-lipoxygenase-activating protein, Alpha-1-acid glycoprotein 1, Protein S100-A9, Perilipin-1, Protein DEK, Protein S100-P, ELMO domain-containing protein 2, Microsomal glutathione S -transferase 1, Phospholipase B-like 1, Cytochrome b-245 heavy chain, Merlin, Complement factor I, Cytochrome b-245 light chain, Casein kinase I isoform gamma-1, Inter-alpha-trypsin inhibitor heavy chain H1, Long- A protein group consisting of chain-fatty-acid--CoA ligase 1 and Inter-alpha-trypsin inhibitor heavy chain H2,
    (E) HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine /threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A-like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo repeat-containing protein 8, Nucleolysin TIA-1 isoform p40, Nucleolysin TIA-1 isoform p40, Nucleolysin TIAR, Integrin beta-3, GMP reductase 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Coactosin-like protein, Integrin alpha-IIb, Protein cereblon, PDZ and LIM domain protein 1, Basic leucine zipper and W2 domain-containing protein 1, LHFPL tetraspan subfamily member 6 protein, WD repeat-containing protein 11, Tyrosine-protein kinase BTK, DENN domain-containing protein 4C, N-acetyl -D-glucosamine kinase, Cofilin-1, Translin-associated protein X, Choline transporter-like protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11, ADP-ribosylation factor 4, FYN-binding protein 1, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D3, Ubiquitin-conjugating enzyme E2 D2, Caveolae-associated protein 2, EH domain-containing protein 3, EH domain-containing protein 1, a group of proteins consisting of cAMP-dependent protein kinase type I-alpha regulatory subunit, synaptotagmin-like protein 4, and charged multivesicular body protein 2a;
    (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, 2'-deoxynucleoside 5 '-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Torsin-1A-interacting protein 1, Annexin A1, Estradiol 17-beta-dehydrogenase 11, Endoplasmic reticulum- Protein group consisting of Golgi intermediate compartment protein 1, Erlin-2, Anoctamin-10, and Ras-related protein Rab-27A, and (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bisphosphate 4-phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptidase, Progressive ankylosis protein homolog, Disintegrin and metalloproteinase domain-containing protein 10, Calpain-7, Choline transporter-like protein 1, LHFPL tetraspan subfamily member 6 protein, STARD3 N-terminal-like protein, Inactive rhomboid protein 2, Serine incorporator 3, Annexin A4, Cas scaffolding protein family member 4, Low-density lipoprotein receptor-related protein 10, Tetraspanin-32, von Willebrand factor A domain-containing protein 5A, Gamma-enolase, MICAL-like protein 2, Vacuolar protein sorting-associated protein 4B, Metalloreductase STEAP3, Choline transporter-like protein 2, Protein transport protein Sec24C, Bis(5'-adenosyl)-triphosphatase ENPP4, Solute carrier family 12 member 4 , Solute carrier family 12 member 7, and a protein group consisting of Solute carrier family 43 member 3,
    A method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma, wherein the amount or concentration of at least one protein selected from the group consisting of is used as an index.
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