Method, kit and biomarker for assisting diagnosis of depression and schizophrenia etc.

Abstract

The present invention addresses the problem of providing: a method, a kit, and a biomarker for assisting the diagnosis of depression or schizophrenia; and a method, a kit, and a biomarker for assisting in the discrimination between depression and bipolar disorder. The present invention provides a method for assisting the diagnosis of depression or schizophrenia, the method including detecting cells that have CD84, as a cell surface antigen, contained in a test sample.

Description

Methods, kits and biomarkers to aid in the diagnosis of depression or schizophrenia

[0001] The present invention relates to a method for assisting in the diagnosis of depression or schizophrenia using cells having CD84 as a cell surface antigen as an indicator, and a method for assisting in the differentiation between depression and bipolar disorder using cells having CD84 as a cell surface antigen as an indicator.The present invention further relates to a kit for assisting in the diagnosis of depression or schizophrenia, and a kit for assisting in the differentiation between depression and bipolar disorder.The present invention further relates to a biomarker for depression or schizophrenia, and a biomarker for assisting in the differentiation between depression and bipolar disorder.

[0002] In modern society, mental illnesses such as depression, bipolar disorder, schizophrenia, and dementia have become a social problem. In particular, depression, characterized by symptoms such as low mood, decreased mental activity, loss of appetite, and insomnia, accounts for a high proportion of mental illnesses. Diagnoses of mental illnesses are often based on subjective information obtained from patients through interviews, but highly accurate diagnoses are difficult. In light of the above, diagnostic methods for mental illnesses using objective indicators (markers, etc.) are being investigated.

[0003] Non-Patent Document 1 describes the detection of depression using cells co-positive for CD33 and CD11b cell surface antigens as an indicator. Non-Patent Document 2 describes the analysis of CD84 gene expression levels in samples from subsyndromal depression (SSD), major depressive disorder (MDD), and healthy subjects, in which red blood cells have been removed from EDTA-clotted peripheral blood, and finds no significant difference in CD84 gene expression levels between patients with major depressive disorder and healthy subjects.

[0004] J. Wei et al. Psychiatry Research 228 (2015) 695-701 Yang C, Hu G, Li Z, Wang Q, Wang X, Yuan C, et al. (2017) PLoS ONE 12(3):e0172692. https: / / doi. org / 10.1371 / journal. bone. 0172692

[0005] An object of the present invention is to provide a method, a kit, and a biomarker for assisting in the diagnosis of depression or schizophrenia.A further object of the present invention is to provide a method, a kit, and a biomarker for assisting in the differentiation between depression and bipolar disorder.

[0006] Chronically stressed mice (chronic social defeat stress mice: CSDS mice) are known to exhibit psychiatric symptoms such as depressive behavior, and are used as a mouse model of psychiatric disorders.

[0007] The present inventors have found that the presence or absence of mental disorders in humans and humans can be diagnosed by separating cell populations from blood cells of CSDS mice or humans by flow cytometry using antibodies against cell surface markers such as CD84, CD33, CD11b, and Ly6G, and then analyzing signals corresponding to the cell count.The present invention was completed based on the above findings.

[0008] The present invention provides the following: <1> A method for assisting in the diagnosis of depression or schizophrenia, comprising detecting cells in a test sample that have CD84 as a cell surface antigen. <2> The method according to <1>, in which the diagnosis of depression or schizophrenia is aided by an indicator comprising the amount of cells that have CD84 as a cell surface antigen. <3> The method according to <2>, in which the indicator comprising the amount of cells that have CD84 as a cell surface antigen is the ratio of the amount of cells that have CD84 as a cell surface antigen or the amount of viable cells that have CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in the test sample. <4> The method according to any one of <1> to <3>, in which the cells further have at least one cell surface antigen selected from CD33, CD11b, and Ly6G. <5> The method according to any one of <1> to <4>, wherein the cells are cells having CD84 and CD33 as cell surface antigens, cells having CD84 and CD11b as cell surface antigens, or cells having CD84, CD33, and CD11b as cell surface antigens. <6> The method according to any one of <1> to <5>, wherein the cells aid in the diagnosis of depression or schizophrenia, but not bipolar disorder. <7> The method according to any one of <1> to <6>, wherein the test sample is a human blood-derived sample, a mouse blood-derived sample, or a mouse spleen-derived sample. <8> The method according to <7>, wherein the test sample is a sample that has been subjected to a procedure to remove red blood cells, or a sample that does not contain red blood cells. <9> A method for aiding in the differentiation between depression and bipolar disorder, comprising detecting cells having CD84 as a cell surface antigen contained in a test sample. <10> The method according to <9>, wherein an indicator comprising the amount of cells having CD84 as a cell surface antigen is used to assist in the differentiation of depression from bipolar disorder. <11> The method according to <10>, wherein the indicator comprising the amount of cells having CD84 as a cell surface antigen is the ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in the test sample.<12> The method according to any one of <9> to <11>, wherein the cells further have at least one cell surface antigen selected from CD33, CD11b, and Ly6G. <13> The method according to any one of <9> to <12>, wherein the cells are cells having CD84 and CD33 on their surface, cells having CD84 and CD11b on their surface, or cells having the cell surface antigens CD84, CD33, and CD11b. <14> A diagnostic support kit for depression or schizophrenia, comprising a substance that binds to CD84. <15> The diagnostic support kit according to <14>, further comprising at least one substance selected from a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G. <16> A kit for supporting the differentiation of depression from bipolar disorder, comprising a substance that binds to CD84. <17> The kit for assisting in the differentiation of <16>, further comprising at least one substance selected from the group consisting of a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G. <18> A biomarker for depression or schizophrenia, comprising cells having CD84 as a cell surface antigen. <19> A biomarker for assisting in the differentiation of depression and bipolar disorder, comprising cells having CD84 as a cell surface antigen.

[0009] According to the present invention, it is possible to accurately assist in the diagnosis of depression or schizophrenia. According to the present invention, it is possible to accurately assist in the differentiation between depression and bipolar disorder.

[0010] FIG. 1 shows the results of separation using a combination of cell surface markers CD11b, CD33, and CD84.

[0011] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0012] <Method for assisting in the diagnosis of depression or schizophrenia; and method for assisting in the differentiation between depression and bipolar disorder> A first aspect of the present invention is a method for assisting in the diagnosis of depression or schizophrenia, comprising detecting cells having CD84 as a cell surface antigen contained in a test sample. The method for assisting in the diagnosis of depression or schizophrenia according to the first aspect of the present invention may also assist in the diagnosis of depression or schizophrenia, but not bipolar disorder. A second aspect of the present invention is a method for assisting in the differentiation between depression and bipolar disorder, comprising detecting cells having CD84 as a cell surface antigen contained in a test sample.

[0013] Depression includes the following: Depressive Disorders: Major Depressive Disorder (MDD), Persistent Depressive Disorder (PDD), Major Mood Dysfunction Disorder (DMDD), Premenstrual Dysphoric Disorder (PMDD), Substance- or Medication-Induced Depressive Disorder, Depressive Disorder Due to Other Medical Illness, and Subsyndromal Depression (SSD). Depressive Disorders are classified in the DSM-5 and include major depressive disorder and other depressive disorders. Subsyndromal depression is not defined in the DSM-5, but includes a state preceding major depression.

[0014] Schizophrenia is a mental illness that causes serious impairments in cognition, emotions, and behavior, and is characterized by the following symptoms: ・Positive symptoms: hallucinations, delusions, disorganized thinking, catatonic behavior, etc. ・Negative symptoms: flat affect, social withdrawal, decreased motivation, anhedonia, etc. ・Cognitive symptoms: decreased attention and concentration, memory impairment, difficulty planning and problem-solving Schizophrenia is classified in the DSM-5, which includes the positive symptoms, negative symptoms, and cognitive symptoms mentioned above. These symptoms may appear in different combinations depending on the individual patient.

[0015] Bipolar disorder is a mental illness characterized by abnormal mood swings, with the following episodes alternating: - Manic episodes: abnormal elation or excitement, excessive energy and activity, overestimation, impulsive behavior, etc. - Depressive episodes: intense sadness or hopelessness, loss of interest or pleasure, fatigue, suicidal thoughts, etc. - Mixed episodes: a state in which manic and depressive symptoms appear simultaneously. Bipolar disorder is classified in the DSM-5 and is divided into bipolar I (with clear manic episodes) and bipolar II (with hypomanic episodes and severe depressive episodes). It also includes rapid cycling (experiencing four or more episodes in a year).

[0016] The test sample may be derived from a test animal, and examples thereof include blood-derived samples such as serum, plasma, whole blood, and buffy coat, as well as biological samples such as urine, saliva, cerebrospinal fluid, semen, tissue fluid, sweat, tears, sputum, mucus, lymph, amniotic fluid, bone marrow fluid, pleural effusion, ascites, indirect fluid, aqueous humor, vitreous humor, bladder washings, and bronchoalveolar lavage fluid. Blood-derived samples such as serum, plasma, and whole blood, urine, or cerebrospinal fluid are preferred, with blood-derived samples such as serum, plasma, and whole blood being more preferred. The test sample is preferably a sample that has been subjected to a procedure to remove red blood cells, or a sample that does not contain red blood cells. Examples of the procedure to remove red blood cells include hemolysis using an erythrocyte lysis buffer (ACK buffer) and centrifugal removal of the supernatant. A sample that has been subjected to a procedure to remove red blood cells may contain trace amounts of red blood cells. Furthermore, a sample that does not contain red blood cells means a sample that is substantially free of red blood cells, and may contain trace amounts of red blood cells.

[0017] The test animals include mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, and rabbits, with humans, monkeys, mice, and rats being preferred, and humans being more preferred.

[0018] The test sample is preferably a human blood-derived sample, a mouse blood-derived sample, or a mouse spleen-derived sample, more preferably a human blood-derived sample or a mouse blood-derived sample, and more preferably human or mouse peripheral blood.

[0019] The condition of the test animal from which the test sample is derived is not particularly limited, and may be an animal suspected of being affected by or at risk of developing depression or schizophrenia, or an animal whose condition or risk of developing depression or schizophrenia is unknown.

[0020] When the test animal is a human, the subject may be, but is not limited to, a person who is suspected of having or being at risk of developing depression or schizophrenia, such as a person who has been diagnosed as possibly having depression or schizophrenia based on diagnostic criteria or other methods, or a person who has been diagnosed as being at risk of developing depression or schizophrenia based on diagnostic criteria or other methods; a person who has not been diagnosed with depression or schizophrenia; a person who has not been diagnosed as possibly having depression or schizophrenia based on diagnostic criteria or other methods; or a person whose condition or risk of developing depression or schizophrenia is unknown, such as a person who has been diagnosed as possibly having depression or schizophrenia based on diagnostic criteria or other methods.

[0021] The test animal may be one suspected of having a mood disorder (depression or bipolar disorder), or when the test animal is a human, the subject may be a subject suspected of having or being at risk of developing a mood disorder, such as a human diagnosed as possibly having a mood disorder based on diagnostic criteria or other methods, or a human diagnosed as being at risk of developing a mood disorder based on diagnostic criteria or other methods.

[0022] The method for obtaining (collecting) a test sample from a test animal is not particularly limited, and may be, for example, by obtaining (collecting) a sample from a test animal based on a method known per se, and may involve separation, concentration, purification, etc., according to a method known per se, if necessary. Furthermore, the test sample may be one immediately after collection from the test animal, or may be a sample that has been preserved after collection. The method for preserving the sample may be any method commonly used in this field.

[0023] In the present invention, cells having CD84 as a cell surface antigen are detected. Detection in the present invention includes qualitative, semi-quantitative, and quantitative methods, with quantitative detection being preferred. Peripheral blood mononuclear cells (PBMCs) are preferred as the cells.

[0024] The cells are more preferably peripheral blood mononuclear cells (PBMCs) having CD84 and at least one cell surface antigen selected from CD33, CD11b, and Ly6G.

[0025] As the cells, cells in which at least one selected from CD3, which distinguishes T cells, B220 and CD19, which distinguish B cells, and CD235a, which distinguishes red blood cells, are not detected as cell surface antigens are preferred, and cells in which CD3, B220, CD19, and CD235a are not detected as cell surface antigens are more preferred.

[0026] CD84, CD33, CD11b, and Ly6G may each have one or more (e.g., 1 to 10, 1 to 5, 1 to 3, or 1 or 2) amino acids deleted, substituted, or added. CD84, CD33, CD11b, and Ly6G may be detected by detecting the protein or by detecting modifications such as sugar chains, with protein detection being preferred.

[0027] Cells having CD84 as a cell surface antigen may also be cells further having at least one cell surface antigen selected from CD33, CD11b, and Ly6G. Cells having CD84 as a cell surface antigen are preferably cells having CD84 and CD33 as cell surface antigens, cells having CD84 and CD11b as cell surface antigens, or cells having CD84, CD33, and CD11b as cell surface antigens, more preferably cells having CD84 and CD33 as cell surface antigens, or cells having CD84, CD33, and CD11b as cell surface antigens, and particularly preferably cells having CD84 and CD33 as cell surface antigens.

[0028] Cells containing CD84 as a cell surface antigen in a test sample can be detected by any method commonly used in the field. Detection can be, for example, detection using a substance that binds to the cell surface antigen CD84, or immunological measurement using an anti-CD84 antibody. Detection can also be performed by flow cytometry. Flow cytometry can include one or both of FSC measurement, which determines cell size based on forward scattered light signals, and SSC measurement, which determines cell granularity based on side scattered light signals.

[0029] When detecting cells having CD84 as a cell surface antigen, a substance that binds to CD84 is used to form a complex between CD84 and the substance that binds to CD84, and the complex is measured to detect cells having CD84 as a cell surface antigen. Specific methods for the above detection may be performed according to methods known per se.

[0030] Examples of substances that bind to CD84 include proteins that bind to CD84 (e.g., antibodies, peptides such as cyclic peptides), nucleic acids that bind to CD84 (e.g., aptamers), and substances that bind to the sugar chains of CD84 (e.g., lectins, anti-sugar chain antibodies), with antibodies that bind to CD84 (i.e., anti-CD84 antibodies) being preferred. The substance that binds to CD84 is preferably one that specifically binds to CD84. Either one or two or more types of substances that bind to CD84 may be used, with the use of only one type being preferred. The substance that binds to CD84 may be a commercially available product or one that is appropriately prepared by conventional methods.

[0031] The anti-CD84 antibody may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination.

[0032] Anti-CD84 antibodies include not only intact immunoglobulin molecules but also fragments thereof, such as Fab and F(ab'), which have the ability to bind to antigens. ２Alternatively, fragment antibodies such as F(ab') and single-chain antibodies (single chain Fv), diabodies, triabodies, tetrabodies, and other synthetic antibodies may be used. The anti-CD84 antibody may be a commercially available antibody, or one prepared according to a method known per se. When preparing an anti-CD84 antibody according to a method known per se, the method may be, for example, that described in "Immunoassay Method" (edited by Biochemical Measurement Research Group, Kodansha, 2014) or JP-A-2018-501202.

[0033] The substance that binds to CD84 may be labeled with a labeling substance, specifically, for example, enzymes such as peroxidase (e.g., horseradish peroxidase (HRP)), microperoxidase, alkaline phosphatase (e.g., bovine small intestinal alkaline phosphatase), and β-galactosidase; ９９ｍ Tc, １３１ I, １２５ I, １４ C. ３ H. ３２ P. ３５ Examples of suitable labeling agents include radioisotopes such as S; fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye, or rhodamine, or derivatives thereof; luminescent substances such as luciferin, luminol, and ruthenium complexes; substances that absorb in the ultraviolet region, such as phenol, naphthol, or anthracene, or derivatives thereof; substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl; dyes such as phycoerythrin (PE), allophycocyanin (APC), HiLyte-based dyes, Alexa-based dyes, and CyDye-based dyes; and nanoparticles such as gold colloids and quantum dots. Among the above, enzymes, fluorescent substances, or dyes are preferred. The method for binding a labeling substance to a substance that binds to CD84 is not particularly limited, and may be performed according to a labeling method known per se. Measurement of these labeling substances may be carried out according to a known measurement method appropriate for the labeling substance.

[0034] A secondary binding substance (e.g., a secondary antibody) that specifically binds to the substance that binds to CD84 may also be used. The secondary binding substance may be labeled with a labeling substance, preferably a labeled substance, and a secondary antibody labeled with a labeling substance is more preferred. The labeling substance and labeling method are the same as those described above, and the preferred methods are also the same.

[0035] Furthermore, labeling with a labeling substance may utilize the binding of avidins and biotins by using a substance that binds to CD84 bound to either avidins or biotins, and a substance that binds to the other of avidins or biotins bound to a labeling substance. Examples of biotins include biotin, iminobiotin, desthiobiotin, biocytin, biotin sulfoxide, etc., with biotin being preferred. Examples of avidins include avidin, tamavidin, tamavidin 2, streptavidin, etc., with streptavidin being preferred. The method of binding avidins or biotins to a substance that binds to CD84 and the method of binding the other of avidins or biotins to a labeling substance may be carried out according to conventional methods.

[0036] The method for assisting in the diagnosis of depression or schizophrenia according to the present invention is a method for distinguishing between healthy subjects, depression, and schizophrenia, or between bipolar disorder, depression, and schizophrenia, and can, for example, perform any of the following: (A) distinguishing between healthy subjects and depression, (B) distinguishing between healthy subjects and schizophrenia, (C) distinguishing between healthy subjects and depression or schizophrenia, (D) distinguishing between depression and schizophrenia, (E) distinguishing between healthy subjects, depression, and schizophrenia, (F) distinguishing between bipolar disorder and depression, (G) distinguishing between bipolar disorder and schizophrenia, (H) distinguishing between bipolar disorder and depression or schizophrenia, or (I) distinguishing between bipolar disorder, depression, and schizophrenia. The above distinctions can be made, for example, by using appropriate reference values ​​(cutoff values). The above differentiation includes the morbidity state and risk of developing the disease to be differentiated. Reference values ​​(cutoff values) will be described later. Assistance in diagnosing depression or schizophrenia, but not bipolar disorder, refers to (F) to (I) above. Note that depression and bipolar disorder are sometimes collectively referred to as mood disorders. In the present invention, the subject may be an animal (e.g., a human) that requires differentiation of a mood disorder (depression or bipolar disorder).

[0037] In the present invention, the diagnosis of depression or schizophrenia may be aided by an indicator including the amount of cells having CD84 as a cell surface antigen. Examples of indicators including the amount of cells having CD84 as a cell surface antigen include the number of the cells, the density of the cells, and the ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in a test sample. Indicators including the amount of cells having CD84 as a cell surface antigen also include measured values ​​that correlate with the number of the cells or the density of the cells, such as absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence amount, luminescence change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, and refractive index change.

[0038] As an indicator including the amount of cells having CD84 as a cell surface antigen, the ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in the test sample can be preferably used.

[0039] That is, as an indicator including the amount of cells having CD84 as a cell surface antigen, preferably, (1) the ratio of the amount of cells having CD84 as a cell surface antigen to the total amount of cells in the test sample; (2) the ratio of the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells in the test sample; (3) the ratio of the amount of cells having CD84 as a cell surface antigen to the total amount of viable cells in the test sample; or (4) the ratio of the amount of viable cells having CD84 as a cell surface antigen to the total amount of viable cells in the test sample can be used, and more preferably, an indicator derived from (2) the ratio of the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells in the test sample, and (4) the ratio of the amount of viable cells having CD84 as a cell surface antigen to the total amount of viable cells in the test sample, and even more preferably, (2) the ratio of the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells in the test sample.

[0040] Specific examples of indicators including the amount of cells having CD84 as a cell surface antigen include: (1-1) the ratio of the amount of cells having CD84 and at least one or more cell surface antigens selected from CD33, CD11b, and Ly6G to the total amount of cells in the test sample; (1-2) the ratio of the amount of cells having CD84 and CD33 as cell surface antigens to the total amount of cells in the test sample; (1-3) the ratio of the amount of cells having CD84 and CD11b as cell surface antigens to the total amount of cells in the test sample; and (1-4) the ratio of the amount of cells having CD84, CD33, and CD1 as cell surface antigens to the total amount of cells in the test sample. (2-1) the proportion of the amount of viable cells further having CD84 and at least one or more cell surface antigens selected from CD33, CD11b, and Ly6G relative to the total amount of cells in the test sample; (2-2) the proportion of the amount of viable cells having CD84 and CD33 as cell surface antigens relative to the total amount of cells in the test sample; (2-3) the proportion of the amount of viable cells having CD84 and CD11b as cell surface antigens relative to the total amount of cells in the test sample; (2-4) the proportion of the amount of viable cells having CD84, CD33, and CD11b as cell surface antigens relative to the total amount of cells in the test sample; (3-1) the ratio of the amount of cells further having CD84 and at least one or more cell surface antigens selected from CD33, CD11b, and Ly6G to the total amount of live cells in the test sample; (3-2) the ratio of the amount of cells having CD84 and CD33 as cell surface antigens to the total amount of live cells in the test sample; (3-3) the ratio of the amount of cells having CD84 and CD11b as cell surface antigens to the total amount of live cells in the test sample; (3-4) the ratio of the amount of CD84, CD33 to the total amount of live cells in the test sample and CD11b as cell surface antigens; (4-1) the proportion of the amount of viable cells further having CD84 and at least one or more cell surface antigens selected from CD33, CD11b, and Ly6G relative to the total amount of viable cells in the test sample; (4-2) the proportion of the amount of viable cells having CD84 and CD33 as cell surface antigens relative to the total amount of viable cells in the test sample; (4-3) the proportion of the amount of viable cells having CD84 and CD11b as cell surface antigens relative to the total amount of viable cells in the test sample;Alternatively, (4-4) the ratio of the amount of viable cells having CD84, CD33, and CD11b as cell surface antigens to the total amount of viable cells in the test sample can be used, preferably (1-2), (1-4), (2-2), (2-4), (3-2), (3-4), (4-2), or (4-4), more preferably (1-2), (2-2), (3-2), or (4-2), and particularly preferably (2-2);

[0041] The ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in a test sample can be determined, for example, by gating on cells using flow cytometry.

[0042] For example, a labeled anti-CD84 antibody is added to a sample containing cells collected from a subject, and the cells are stained to prepare a flow cytometry sample. The total cell amount in the test sample can be determined by measuring the cell amount in the sample. Next, by gating on CD84-positive cells using flow cytometry, a cell population having CD84 as a cell surface antigen can be isolated. The amount of cells having CD84 as a cell surface antigen can be determined by measuring the cell amount of the isolated cell population having CD84 as a cell surface antigen.

[0043] When measuring the total viable cell amount in a test sample, for example, 7-AAD(-) viable cells are gated on a sample containing cells collected from a subject using 7-AAD, a marker that distinguishes between viable and dead cells. The total viable cell amount in the test sample can be determined by measuring the cell amount of the separated 7-AAD(-) viable cell group. Next, for the separated 7-AAD(-) viable cell group, flow cytometry is used to gate on CD84-positive cells, thereby isolating a viable cell group having CD84 as a cell surface antigen. The amount of viable cells having CD84 as a cell surface antigen can be determined by measuring the cell amount of the separated viable cell group having CD84 as a cell surface antigen.

[0044] Cells further having at least one cell surface antigen selected from CD33, CD11b, and Ly6G, cells having CD84 and CD33 on their surface, cells having CD84 and CD11b on their surface, or cells having CD84, CD33, and CD11b on their surface can be detected by any method commonly used in this field, specifically, for example, immunological assays such as flow cytometry analysis. Details of immunological assays and flow cytometry analysis are as described above.

[0045] When detecting cells that further have at least one cell surface antigen selected from CD33, CD11b, and Ly6G, cells that have CD84 and CD33 on their surface, cells that have CD84 and CD11b on their surface, or cells that have CD84, CD33, and CD11b on their surface, a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G may be used.

[0046] Details of the substance that binds to CD33, the substance that binds to CD11b, and the substance that binds to Ly6G, as well as the case of labeling them with a labeling substance, are as described for the substance that binds to CD84.

[0047] An index including the amount of cells having CD84 as a cell surface antigen may be used to assist in the diagnosis of depression or schizophrenia, or to assist in the differentiation between depression and bipolar disorder, by combining values ​​obtained by combining two or more types of amounts of cells having CD84 as a cell surface antigen.

[0048] Furthermore, an indicator including the amount of cells having CD84 as a cell surface antigen may be used to aid in the diagnosis of depression or schizophrenia or to aid in the differentiation between depression and bipolar disorder by combining, in addition to the amount of cells having CD84 as a cell surface antigen, a value obtained by detecting a biomarker for the disease to be differentiated other than cells having CD84 as a cell surface antigen contained in the test sample (preferably, the amount of a biomarker for the disease to be differentiated other than cells having CD84 as a cell surface antigen). Examples of biomarkers for the disease to be differentiated other than cells having CD84 as a cell surface antigen include tryptophan metabolites such as serotonin based on the monoamine hypothesis of depression, noradrenaline, dopamine, inflammatory cytokines (IL-1β, IL-6, TNF-α, etc.) based on the inflammatory hypothesis, cortisol, ACTH, DHEA-S based on the HPA axis, and BDNF based on the neuroplasticity hypothesis. Methods for obtaining the above-mentioned combined value include, for example, multivariate analysis such as multiple logistic regression analysis, discriminant analysis, Poisson regression analysis, multiple regression analysis, Cox's proportional hazards model, path analysis, etc. Hereinafter, the above-mentioned combined value and the index containing the amount of cells having CD84 as a cell surface antigen may be abbreviated as a value derived from the test sample.

[0049] In a first aspect of the present invention, the method may include assisting in the diagnosis of depression or schizophrenia based on the results of detecting cells having CD84 as a cell surface antigen contained in a test sample. In a second aspect of the present invention, the method may include assisting in the differentiation of depression from bipolar disorder based on the detection of cells having CD84 as a cell surface antigen contained in a test sample. The above diagnosis and differentiation include the morbidity state and risk of developing the target disease.

[0050] For example, the diagnosis of depression or schizophrenia may be aided by comparing the magnitude relationship between the value derived from the test sample and a preset reference value (such as a cutoff value).Similarly, the differentiation between depression and bipolar disorder may be aided by comparing the magnitude relationship between the value derived from the test sample and a preset reference value (such as a cutoff value).

[0051] The method for determining the reference value (cutoff value) is not particularly limited, and can be determined for each subject to be differentiated by, for example, measuring the amount of cells having CD84 as a cell surface antigen contained in a test sample derived from an animal suffering from depression or schizophrenia, and the amount of cells having CD84 as a cell surface antigen contained in a test sample derived from a healthy animal or an animal suffering from bipolar disorder, and then using the obtained amounts of each cell, a reference value can be determined for each subject to be differentiated by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis).

[0052] In setting the reference value (cutoff value), it is preferable to take into consideration sensitivity, specificity, positive predictive value, negative predictive value, etc. The reference value (cutoff value) can be set, for example, so that the sensitivity is 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more; for example, it can be set so that the specificity is 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0053] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggests that "the test animal may have depression, may have schizophrenia, may have depression or schizophrenia, or the test animal is likely to have depression, schizophrenia, or depression or schizophrenia," or the like; on the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggests "the test animal is not likely to have depression, is not likely to have schizophrenia, is not likely to have depression or schizophrenia, or is unlikely to have depression, is unlikely to have schizophrenia, or is unlikely to have depression or schizophrenia," or the like.

[0054] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggest that "the test animal is not likely to have bipolar disorder, or is unlikely to have bipolar disorder" and "the test animal is likely to have depression, schizophrenia, depression or schizophrenia, or is likely to have depression, schizophrenia, or depression or schizophrenia." On the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggest "the test animal is not likely to have bipolar disorder, or is unlikely to have bipolar disorder" and "the test animal is not likely to have depression, is not likely to have schizophrenia, is not likely to have depression or schizophrenia, or is unlikely to have depression, is unlikely to have schizophrenia, or is unlikely to have depression or schizophrenia."

[0055] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggests that "the test animal may have depression rather than bipolar disorder, or that the test animal is likely to have depression rather than bipolar disorder," or on the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggests "the test animal is unlikely to have depression rather than bipolar disorder, or that the test animal is unlikely to have depression rather than bipolar disorder," or

[0056] When data suggesting that a subject may have depression or schizophrenia is obtained through the present invention, a physician may conduct further tests such as a medical interview, psychological tests, blood tests, genetic tests, electroencephalograms, and imaging tests (MRI, PET, etc.), and can diagnose the subject as having depression or schizophrenia based on the results of these tests.

[0057] When data suggesting that a subject may have depression rather than bipolar disorder is obtained through the present invention, a physician may conduct further tests such as a medical interview, psychological tests, blood tests, genetic tests, electroencephalograms, and imaging tests (MRI, PET, etc.), and can diagnose the subject as having depression by taking these results into consideration.

[0058] <Specific examples of a method for assisting in the diagnosis of depression or schizophrenia and a method for assisting in the differentiation between depression and bipolar disorder> Specific examples of a method for assisting in the diagnosis of depression or schizophrenia and a method for assisting in the differentiation between depression and bipolar disorder are described below. Blood collected from a subject animal (e.g., a human) in an EDTA2Na blood collection tube is diluted with ACK buffer (0.15 M NH ４ Cl, 0.1M KHCO ３ , 0.0001M EDTA 2Na) or similar is added to lyse red blood cells. Centrifuge at 100-2000g for 1-15 minutes, remove the supernatant, and add PBS. Centrifuge at 100-2000g for 1-15 minutes and remove the supernatant. Add Fc Blocking Reagent human to the resulting cell pellet, incubate at room temperature for 1-15 minutes, then add PBS. Centrifuge at 100-2000g for 1-15 minutes and remove the supernatant. Add a solution containing labeled CD84 antibody to the cell pellet and incubate for 5 minutes to 1 hour to stain the cells. After incubation, add PBS and centrifuge at 100-1500g for 1-15 minutes and remove the supernatant. Add a BSA / PBS solution containing 7-AAD to the cell pellet to suspend it and use it as a sample for flow cytometry. Using the above sample, a cell population expressing CD84 is separated by flow cytometry, and the number of viable cells is measured. Specifically, first, 7-AAD (-) viable cells are gated using 7-AAD, a marker for distinguishing between live and dead cells, and then CD84-positive cells are gated, and the number of viable cells in each separated cell population is measured.

[0059] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggests that "the test animal may have depression, may have schizophrenia, may have depression or schizophrenia, or the test animal is likely to have depression, schizophrenia, or depression or schizophrenia," or the like; on the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggests "the test animal is not likely to have depression, is not likely to have schizophrenia, is not likely to have depression or schizophrenia, or is unlikely to have depression, is unlikely to have schizophrenia, or is unlikely to have depression or schizophrenia," or the like.

[0060] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggest that "the test animal is not likely to have bipolar disorder, or is unlikely to have bipolar disorder" and "the test animal is likely to have depression, schizophrenia, depression or schizophrenia, or is likely to have depression, schizophrenia, or depression or schizophrenia." On the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggest "the test animal is not likely to have bipolar disorder, or is unlikely to have bipolar disorder" and "the test animal is not likely to have depression, is not likely to have schizophrenia, is not likely to have depression or schizophrenia, or is unlikely to have depression, is unlikely to have schizophrenia, or is unlikely to have depression or schizophrenia."

[0061] As a result of comparing the magnitude with the above-mentioned reference value (cutoff value, etc.), for example, (i) if the value derived from the test sample is equal to or greater than a predetermined reference value (cutoff value, etc.), data may be obtained that suggests that "the test animal may have depression rather than bipolar disorder, or that the test animal is likely to have depression rather than bipolar disorder," or on the other hand, (ii) if the value derived from the test animal is less than or equal to a predetermined reference value (cutoff value, etc.), data may be obtained that suggests "the test animal is unlikely to have depression rather than bipolar disorder, or that the test animal is unlikely to have depression rather than bipolar disorder," or

[0062] <Method for Obtaining Data to Assist in the Diagnosis of Depression or Schizophrenia, and Method for Obtaining Data to Assist in the Differentiation Between Depression and Bipolar Disorder> The method for obtaining data to assist in the diagnosis of depression or schizophrenia of the present invention comprises detecting cells having CD84 as a cell surface antigen in a test sample, and obtaining data to assist in the diagnosis of depression or schizophrenia of the present invention. The method for obtaining data to assist in the differentiation between depression and bipolar disorder of the present invention comprises detecting cells having CD84 as a cell surface antigen in a test sample, and obtaining data to assist in the differentiation between depression and bipolar disorder of the present invention.

[0063] The data obtained in the method of the present invention includes: (i) a result obtained by detecting cells having CD84 as a cell surface antigen contained in a test sample (preferably, an indicator including the amount of cells having CD84 as a cell surface antigen); and (ii) (iii) a value obtained by combining two or more types of amounts of cells having CD84 as a cell surface antigen, or a value obtained by combining the amount of cells having CD84 as a cell surface antigen and a value obtained by detecting a biomarker related to the disease to be differentiated other than cells having CD84 as a cell surface antigen that is contained in the test sample (preferably, the amount of a biomarker related to the disease to be differentiated other than cells having CD84 as a cell surface antigen), and which is obtained by subjecting the test sample to multivariate analysis such as multiple logistic regression analysis, discriminant analysis, Poisson regression analysis, multiple regression analysis, Cox's proportional hazards model, path analysis, etc.; (iv) data showing the magnitude relationship between the result of (i) above (preferably, an index including the amount of cells having CD84 as a cell surface antigen) or the value obtained in (ii) above and the aforementioned reference value (cutoff value, etc.); If the value is equal to or greater than a predetermined standard value (cutoff value, etc.), the data may suggest that "the subject animal may have depression, may have schizophrenia, may have depression or schizophrenia, or the subject animal is likely to have depression, may have schizophrenia, or is likely to have depression or schizophrenia," "there is no possibility of bipolar disorder, or the possibility of bipolar disorder is low," and "the subject animal may have depression, may have schizophrenia, may have depression or schizophrenia, or the subject animal is likely to have depression, may have schizophrenia, or is likely to have depression or schizophrenia," "the subject animal may have depression and not bipolar disorder, or the subject animal is likely to have depression and not bipolar disorder," etc. (similar to data suggested as a result of comparing the magnitude with the standard value (cutoff value, etc.) in the above-mentioned "Method for assisting in the diagnosis of depression or schizophrenia; and method for assisting in the differentiation between depression and bipolar disorder").

[0064] The "detection of cells having CD84 as a cell surface antigen contained in a test sample" in the method for obtaining data of the present invention is as described above in this specification, and preferred examples, specific examples, etc. are also the same.

[0065] <Method for assisting in the diagnosis of depression or schizophrenia and treating depression or schizophrenia, and method for assisting in the differentiation between depression and bipolar disorder and treating depression> The method for assisting in the diagnosis of depression or schizophrenia and treating depression or schizophrenia according to the present invention includes administering appropriate treatment to a patient from whom data suggesting the possibility of depression or schizophrenia has been obtained according to the present invention. It also includes administering appropriate treatment to a patient from whom data suggesting the possibility of depression rather than bipolar disorder has been obtained according to the present invention.

[0066] Appropriate treatments for depression include psychotherapy, including interviews with a doctor or other medical professional, drug therapy using antidepressants such as amoxapine, maprotiline, fluvoxamine, paroxetine, milnacipran, and mirtazapine, modified electroconvulsive therapy, family therapy, and rehabilitation.

[0067] Appropriate treatments for schizophrenia include psychosocial therapy, such as interviews with a doctor or other medical professional, drug therapy by administering typical psychotropic drugs such as chlorpromazine, haloperidol, and fluphenazine, atypical antipsychotic drugs such as aripiprazole, olanzapine, risperidone, lurasidone, and clozapine, antidepressants, and anti-anxiety drugs, modified electroconvulsive therapy, family therapy, and rehabilitation.

[0068] <Diagnostic aid kit for depression or schizophrenia: and diagnostic aid kit for differentiating depression from bipolar disorder> The present invention relates to a diagnostic aid kit for depression or schizophrenia, which comprises a substance that binds to CD84. The diagnostic aid kit of the present invention may further comprise at least one substance selected from the group consisting of a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G.

[0069] The present invention further relates to a kit for assisting in the differentiation between depression and bipolar disorder, comprising a substance that binds to CD84. The kit of the present invention may further comprise at least one substance selected from the group consisting of a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G.

[0070] The diagnostic aid kit for depression or schizophrenia of the present invention and the diagnostic aid kit for differentiation between depression and bipolar disorder of the present invention are collectively referred to as the kit of the present invention.

[0071] In the kit of the present invention, the substance that binds to CD84 may be in a solution state, or in a frozen state, dry state, or lyophilized state. When the kit of the present invention further comprises at least one substance selected from a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G, the substance that binds to CD33, the substance that binds to CD11b, and the substance that binds to Ly6G may each be in a solution state, or in a frozen state, dry state, or lyophilized state.

[0072] The substance that binds to CD84, the substance that binds to CD33, the substance that binds to CD11b, and the substance that binds to Ly6G are as explained in <Method for assisting in the diagnosis of depression or schizophrenia; and method for assisting in the differentiation between depression and bipolar disorder>, and preferred examples, specific examples, etc. are also the same.

[0073] The kit of the present invention may further comprise a secondary binding substance (e.g., a secondary antibody) that specifically binds to the substance that binds to CD84. The secondary binding substance in the kit of the present invention is the same as that described above in the method of the present invention, and preferred examples thereof are also the same. The secondary binding substance in the kit of the present invention may be labeled with a labeling substance, and the labeling substance and labeling method are the same as those described above in the method of the present invention, and preferred examples thereof are also the same.

[0074] The substance that binds to CD84 in the kit of the present invention may be one to which either avidin or biotin is bound. The avidin and biotin are the same as those described above in the method of the present invention, and preferred examples thereof are also the same. The kit of the present invention may further include a labeled substance to which either avidin or biotin is bound. The labeled substance and labeling method are the same as those described above in the method of the present invention, and preferred examples thereof are also the same.

[0075] The concentration (amount) of the substance that binds to CD84 in the kit of the present invention may be appropriately set within a range typically used in this field depending on the measurement method. For example, the substance that binds to CD84 is preferably contained in an amount that results in a concentration of 10 to 20,000 ng / mL, or 100 to 10,000 ng / mL, at the time of use.

[0076] In the kit of the present invention, reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc. may be present together with the substance that binds to CD84. The concentrations and pH of these reagents may also be appropriately selected from the ranges commonly used in this field.

[0077] The kit of the present invention may contain, in addition to the substance that binds to CD84, a reagent for detecting CD84-bearing cells. Examples of such a reagent include a detergent, a sample diluent (a reagent for diluting a sample), and a reagent for detecting a labeled substance. The concentration, pH, and other parameters of these reagents may be appropriately selected from ranges commonly used in this field.

[0078] When an antibody is labeled with a labeling substance, the reagent for detecting the labeling substance detects the label in the antibody labeled with the labeling substance, and examples thereof include substrates for absorbance measurement such as tetramethylbenzidine and orthophenylenediamine, fluorescent substrates such as hydroxyphenylpropionic acid and hydroxyphenylacetic acid, and luminescent substances such as luminol, as well as reagents for absorbance measurement such as 4-nitrophenyl phosphate, and fluorescent substrates such as 4-methylumbelliferyl phosphate.

[0079] Furthermore, the kit of the present invention may contain instructions. The instructions refer to instructions for use with the reagent of the present invention, package inserts, pamphlets (leaflets), etc., which substantially describe in text and / or diagrams the characteristics, principles, operating procedures, and evaluation procedures of the method of the present invention for assisting in the diagnosis of depression or schizophrenia and the method of the present invention for assisting in the differentiation of depression from bipolar disorder. These instructions may be written in multiple documents or may be written together in a single document.

[0080] According to the kit of the present invention, the method of the present invention for assisting in the diagnosis of depression or schizophrenia, and the method of the present invention for assisting in the differentiation between depression and bipolar disorder can be carried out simply, quickly, and accurately.

[0081] <Biomarker for depression or schizophrenia; and biomarker to aid in the differentiation between depression and bipolar disorder> The present invention relates to a biomarker for depression or schizophrenia, comprising cells having CD84 as a cell surface antigen. The present invention further relates to a biomarker to aid in the differentiation between depression and bipolar disorder, comprising cells having CD84 as a cell surface antigen. Depression, schizophrenia, and bipolar disorder in the markers of the present invention are as described above in this specification.

[0082] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the examples, M represents mol / L.

[0083] Example 1: Differentiation of healthy subjects and patients with depression by flow cytometry using cell surface markers The reagents used in the following experiments are as follows: Fc Blocking Reagent human: Miltenyi Biotec (130-059-901) PE-Cy7-labeled CD11b antibody: BioLegenD (M1 / 70) APC-labeled CD33 antibody: BioLegenD (WM53) PE-labeled CD84 antibody: BioLegenD (CD84.1.21)

[0084] <1-1> Sample Preparation: 600 μL of blood collected from 10 healthy subjects and 5 patients with depression (major depressive disorder (MDD)) in EDTA2Na blood collection tubes was added to ACK buffer (0.15 M NH ４ Cl, 0.1M KHCO ３ 19 mL of 0.0001 M EDTA 2Na was added to each well and incubated at room temperature for 5 minutes to lyse red blood cells. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 5 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and 50 μL of Fc Blocking Reagent human was added to the cell pellet. After incubation at room temperature for 5 minutes, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and antibody solutions (1% BSA / PBS, PE-Cy7-labeled CD11b antibody, APC-labeled CD33 antibody, PE-labeled CD84 antibody) were added to the cell pellet and incubated at 4°C for 20 minutes to stain the cells. After incubation, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and the cell pellet was suspended in 1% BSA / PBS solution containing 7-Amino-Actinomycin D (7-AAD) to prepare a sample for flow cytometry.

[0085] <1-2> Measurement of cell counts in each cell group by flow cytometry Using each sample treated by the method described in <1-1>, cell groups expressing the surface antigens listed in Table 1 below (cell groups of Types 0 to 4) were separated by flow cytometry, and the number of viable cells was measured. Specifically, 7-AAD (-) viable cells were first gated using 7-AAD, a marker for distinguishing between live and dead cells.

[0086] Next, in the case of a cell population expressing the surface antigens CD11b, CD33, and CD84 of Type 4, a cell population positive for CD11b and CD33 was gated from the sample in which the viable 7-AAD(-) cells were gated. Next, a cell population positive for CD84 was gated from the cell population positive for CD11b and CD33, and the number of viable cells in the resulting cell population expressing the surface antigens CD11b, CD33, and CD84 (the number of viable Type 4 cells) was measured.

[0087] Using a similar method, cell groups expressing the surface antigens listed in Table 1 below (Type 0 to Type 3) were gated from samples obtained by gating on viable 7-AAD(-) cells, and the number of viable cells in each of the Type 0 to Type 3 cell groups was measured. Statistical analysis (t-test) was performed between healthy subjects and patients with depression, and p-values ​​were calculated. The results are shown in Table 1.

[0088] Furthermore, the number of all cells (total cell count) in the samples treated by method <1-1> was measured by flow cytometry. The viable cell count / total cell count (% Total) for each of the cell groups Type 0 to Type 4 was calculated. Statistical analysis (t-test) was also performed between healthy subjects and patients with depression, and p-values ​​were calculated. The results are shown in Table 2. Furthermore, the results of separating cell groups expressing the surface antigens of the cell surface markers CD11b, CD33, and CD84 are shown in Figure 1.

[0089] <1-3> Results As shown in the p-values ​​in Table 1, it was found that the cell group expressing CD84 (Type 1) can distinguish between healthy subjects and patients with depression with higher accuracy than the cell group expressing CD11b and CD33 (Type 0). Furthermore, it was found that the cell group expressing CD84 and CD11b (Type 2), the cell group expressing CD84 and CD33 (Type 3), and the cell group expressing CD84, CD11b, and CD33 (Type 4) can distinguish between healthy subjects and patients with depression with even higher accuracy.

[0090]

[0091] As shown in the p-values ​​in Table 2, it was found that the cell group expressing CD84 (Type 1) can distinguish between healthy subjects and patients with depression with higher accuracy than the cell group expressing CD11b and CD33 (Type 0). Furthermore, it was found that the cell group expressing CD84 and CD11b (Type 2), the cell group expressing CD84 and CD33 (Type 3), and the cell group expressing CD84, CD11b, and CD33 (Type 4) can distinguish between healthy subjects and patients with depression with even higher accuracy.

[0092]

[0093] Example 2: Differentiation of healthy subjects and schizophrenic patients by flow cytometry using cell surface markers <2-1> Sample preparation 600 μL of blood collected from 10 healthy subjects and 15 schizophrenic patients in EDTA2Na blood collection tubes was diluted with ACK buffer (0.15 M NH ４ Cl, 0.1M KHCO ３ 19 mL of 0.0001 M EDTA 2Na was added to each well and incubated at room temperature for 5 minutes to lyse red blood cells. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 5 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and 50 μL of Fc Blocking Reagent human was added to the cell pellet. After incubation at room temperature for 5 minutes, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and antibody solutions (1% BSA / PBS, PE-Cy7-labeled CD11b antibody, APC-labeled CD33 antibody, PE-labeled CD84 antibody) were added to the cell pellet and incubated at 4°C for 20 minutes to stain the cells. After incubation, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and the cell pellet was suspended in a 1% BSA / PBS solution containing 7-AAD to prepare a sample for flow cytometry.

[0094] <2-2> Separation of each cell population by flow cytometry Using each sample treated by the method in <2-1>, cell populations expressing the surface antigens listed in Table 3 below (cell populations of Types 1 to 3) were separated by flow cytometry, and the viable cell counts were measured. Specifically, 7-AAD (-) viable cells were first gated using 7-AAD, a marker for distinguishing between viable and dead cells.

[0095] Next, in the case of gating on the Type 3 cell population expressing the CD11b, CD33, and CD84 surface antigens, the CD11b- and CD33-positive cell population was gated from the sample in which the 7-AAD(-) live cells were gated. Next, the CD84-positive cell population was gated from the CD11b- and CD33-positive cell population, and the number of cells in the cell population expressing the CD11b, CD33, and CD84 surface antigens (the number of Type 3 cells) was measured.

[0096] Using a similar method, each cell group (Type 1 to Type 3) expressing the surface antigens listed in Table 3 below was gated from the sample obtained by gating on live 7-AAD(-) cells, and the number of live cells in each cell group of Type 1 to Type 3 was measured. Statistical analysis (t-test) was performed on healthy subjects and schizophrenia patients, and p-values ​​were calculated. The results are shown in Table 3.

[0097] Furthermore, the number of all cells (total cell count) in the samples treated by the method of <2-1> was measured by flow cytometry. The results are shown in Table 4 below.

[0098] <2-3> Results As shown in the p-values ​​in Table 3, it was found that healthy individuals and schizophrenic patients can be differentiated based on the cell group expressing CD84 and CD11b (Type 1), the cell group expressing CD84 and CD33 (Type 2), and the cell group expressing CD84, CD11b, and CD33 (Type 3).

[0099]

[0100] As shown in the p-values ​​in Table 4, it was found that healthy individuals can be distinguished from schizophrenic patients based on the cell group expressing CD84 and CD11b (Type 1), the cell group expressing CD84 and CD33 (Type 2), and the cell group expressing CD84, CD11b, and CD33 (Type 3).

[0101]

[0102] Example 3: Differentiation of mood disorders (patients with depression and bipolar disorder) by flow cytometry using cell surface markers <3-1> Sample preparation 600 μL of blood collected from five patients with depression (major depressive disorder (MDD)) and four patients with bipolar disorder in EDTA2Na blood collection tubes was diluted with ACK buffer (0.15 M NH ４ Cl, 0.1M KHCO ３ 19 mL of 0.0001 M EDTA 2Na was added to each well and incubated at room temperature for 5 minutes to lyse red blood cells. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 5 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and 50 μL of Fc Blocking Reagent human was added to the cell pellet. After incubation at room temperature for 5 minutes, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and antibody solutions (1% BSA / PBS, PE-Cy7-labeled CD11b antibody, APC-labeled CD33 antibody, PE-labeled CD84 antibody) were added to the cell pellet and incubated at 4°C for 20 minutes to stain the cells. After incubation, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and the cell pellet was suspended in a 1% BSA / PBS solution containing 7-AAD to prepare a sample for flow cytometry.

[0103] <3-2> Separation of Each Cell Group by Flow Cytometry Using each sample treated by the method of <3-1>, cell groups expressing the surface antigens listed in Table 5 below (cell groups of Type 1 to Type 4) were separated by flow cytometry, and the number of viable cells was measured. Specifically, first, live 7-AAD(-) cells were gated using 7-AAD, a marker that distinguishes between live and dead cells. Next, in the case of a cell group expressing the surface antigens CD11b, CD33, and CD84 of Type 4, a cell group positive for CD11b and CD33 was gated from the sample gated on the live 7-AAD(-) cells. Next, a cell group positive for CD84 was gated from the cell group positive for CD11b and CD33, and the number of cells in the cell group expressing the surface antigens CD11b, CD33, and CD84 (cell number of Type 4) was measured.

[0104] Using a similar method, each cell group (Type 1 to Type 4) expressing the surface antigens listed in Table 5 below was gated from the sample obtained by gating on 7-AAD(-) viable cells, and the number of viable cells in each of the cell groups Type 1 to Type 3 was measured. Statistical analysis (t-test) was performed between healthy subjects and patients with depression, and p-values ​​were calculated. The results are shown in Table 5.

[0105] In addition, the number of all cells (total cell count) in the samples treated by method <3-1> was measured by flow cytometry. The viable cell count / total cell count (% Total) of each of the cell groups of Type 1 to Type 4 was calculated. Statistical analysis (t-test) was also performed between patients with depression and patients with bipolar disorder, and p-values ​​were calculated. The results are shown in Table 6 below.

[0106] <3-3> Results As shown in the p-values ​​in Table 5, it was found that the cell group expressing CD84 (Type 1) can distinguish between patients with depression and patients with bipolar disorder. Furthermore, it was found that the cell group expressing CD84 and CD11b (Type 2), the cell group expressing CD84 and CD33 (Type 3), and the cell group expressing CD84, CD11b, and CD33 (Type 4) can distinguish between patients with depression and patients with bipolar disorder with even higher accuracy.

[0107]

[0108] As shown by the p-values ​​in Table 6, it was found that the CD84-expressing cell group (Type 1) can distinguish between patients with depression and patients with bipolar disorder. Furthermore, it was found that the CD84 and CD11b-expressing cell group (Type 2), the CD84 and CD33-expressing cell group (Type 3), and the CD84, CD11b, and CD33-expressing cell group (Type 4) can distinguish between patients with depression and patients with bipolar disorder with even higher accuracy.

[0109]

[0110] Example 4: Differentiation of healthy and CSDS mice by flow cytometry using cell surface markers The reagents used in the following experiments are as follows: Fc Blocking Reagent: Biolegend (101301) BV-510 labeled Ly6G antibody: Biolegend (1A8) PE-Cy7 labeled CD11b antibody: BioLegenD (M1 / 70) APC labeled CD33 antibody: BioLegenD (W18124D) PE labeled CD84 antibody: BioLegenD (mCD84.7)

[0111] <4-1> Sample Preparation: 600 μL of blood collected from three healthy mice and three CSDS mice in EDTA2Na blood collection tubes was added to ACK buffer (0.15 M NH ４ Cl, 0.1M KHCO ３ 19 mL of 0.0001 M EDTA 2Na was added to each well and incubated at room temperature for 5 minutes to lyse red blood cells. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 5 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 50 μL of Fc Blocking Reagent was added to the cell pellet. After incubation at room temperature for 5 minutes, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and antibody solutions (1% BSA / PBS, VB510-labeled Ly6G antibody, PE-Cy7-labeled CD11b antibody, APC-labeled CD33 antibody, PE-labeled CD84 antibody) were added to the cell pellet and incubated at 4°C for 20 minutes to stain the cells. After incubation, 1 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature to remove the supernatant, and the cell pellet was suspended in a 1% BSA / PBS solution containing 7-AAD to prepare a sample for flow cytometry.

[0112] <4-2> Separation of Novel Cell Populations by Flow Cytometry Using each sample treated by the method described in <4-1>, cell populations expressing the surface antigens listed in Table 7 below (Type 0 to Type 2 cell populations) were separated by flow cytometry, and the number of viable cells was measured. Specifically, 7-AAD(-) viable cells were first gated using 7-AAD, a marker for distinguishing between live and dead cells. Next, in the case of Type 2 cell populations expressing CD33 and CD84 surface antigens, CD33-positive cell populations were gated from the sample gated on the 7-AAD(-) viable cells. Next, CD84-positive cell populations were gated from the CD33-positive cell populations, and the number of cells in the cell populations expressing CD33 and CD84 surface antigens (Type 2 cell count) was measured.

[0113] Using a similar method, cell groups (Type 0 to Type 2) expressing the surface antigens listed in Table 1 below were gated from samples obtained by gating on live 7-AAD(-) cells, and the number of live cells in each of the Type 0 to Type 2 cell groups was measured. Statistical analysis (t-test) was performed between healthy mice and CSDS mice, and p-values ​​were calculated. The results are shown in Table 7.

[0114] <4-3> Results As shown in the p-values ​​in Table 7, it was found that healthy mice and CSDS mice could be distinguished from each other based on the cell group (Type 1) in which CD84 and Ly6G were expressed.

[0115]

[0116] In addition, the number of all cells (total cell count) in the samples treated by method <4-1> was measured by flow cytometry. (Number of viable cells in the Type 0 to Type 2 cell groups) / Total number of viable cells (% Total) was calculated. Statistical analysis (t-test) was also performed between healthy mice and CSDS mice to calculate p-values. The results are shown in Table 8 below.

[0117] <4-3> Results As shown in the p-values ​​in Table 8, it was found that the cell group expressing CD84 and Ly6G (Type 1) and the cell group expressing CD84 and CD33 (Type 2) could distinguish healthy mice from CSDS mice with higher accuracy than the cell group expressing CD11b and CD33 (Type 0).

[0118]

Claims

1. A method for assisting in the diagnosis of depression or schizophrenia, comprising detecting cells having CD84 as a cell surface antigen contained in a test sample.

2. The method according to claim 1, wherein the diagnosis of depression or schizophrenia is aided by an index comprising the amount of cells having CD84 as a cell surface antigen.

3. The method according to claim 2, wherein the indicator comprising the amount of cells having CD84 as a cell surface antigen is the ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in the test sample.

4. The method according to claim 1 or 2, wherein the cells further have at least one cell surface antigen selected from CD33, CD11b, and Ly6G.

5. The method of claim 1 or 2, wherein the cells are cells having CD84 and CD33 as cell surface antigens, cells having CD84 and CD11b as cell surface antigens, or cells having CD84, CD33 and CD11b as cell surface antigens.

6. The method of claim 1 or 2, which aids in the diagnosis of depression or schizophrenia, but not bipolar disorder.

7. The method according to claim 1 or 2, wherein the test sample is a human blood-derived sample, a mouse blood-derived sample, or a mouse spleen-derived sample.

8. The method according to claim 7, wherein the test sample is a sample that has been subjected to a procedure to remove red blood cells or a sample that does not contain red blood cells.

9. A method for assisting in the differentiation between depression and bipolar disorder, comprising detecting cells having CD84 as a cell surface antigen contained in a test sample.

10. The method according to claim 9, wherein the differentiation between depression and bipolar disorder is aided by an indicator comprising the amount of cells having CD84 as a cell surface antigen.

11. The method according to claim 10, wherein the indicator comprising the amount of cells having CD84 as a cell surface antigen is the ratio of the amount of cells having CD84 as a cell surface antigen or the amount of viable cells having CD84 as a cell surface antigen to the total amount of cells or the total amount of viable cells in the test sample.

12. The method according to claim 9 or 10, wherein the cells further have at least one cell surface antigen selected from CD33, CD11b, and Ly6G.

13. The method of claim 9 or 10, wherein the cells are cells having CD84 and CD33 on their surface, cells having CD84 and CD11b on their surface, or cells having CD84, CD33 and CD11b cell surface antigens.

14. A diagnostic aid kit for depression or schizophrenia, comprising a substance that binds to CD84.

15. The diagnostic auxiliary kit according to claim 14, further comprising at least one substance selected from the group consisting of a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G.

16. An aid kit for distinguishing between depression and bipolar disorder, comprising a substance that binds to CD84.

17. The differentiation aid kit according to claim 16, further comprising at least one substance selected from the group consisting of a substance that binds to CD33, a substance that binds to CD11b, and a substance that binds to Ly6G.

18. A biomarker for depression or schizophrenia, comprising cells having CD84 as a cell surface antigen.

19. An auxiliary biomarker for distinguishing between depression and bipolar disorder, including cells that have CD84 as a cell surface antigen.

Images