JP2012018160A - Diagnostic method and diagnostic reagent kit for psychiatric disorder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diagnostic reagent kit and a diagnostic method which enable the extracorporeal diagnosis of a psychiatric disorder.SOLUTION: The diagnostic reagent kit of the present invention can determine the presence or absence and the extent of a mental disorder by measuring the Shati content of blood or urine of a human or an animal as a subject. The diagnostic reagent kit can also determine whether or not the subject has contracted a mental related illness such as drug addiction, schizophrenia, and depression, and the progression of the illness. Consequently, the diagnostic reagent kit is useful for treatment of a mental related illness such as drug addiction, schizophrenia, and depression.

Description

The present invention relates to a method for diagnosing a mental disorder characterized by using an antibody against a mental disorder-related protein Shati and a diagnostic kit for a mental disorder characterized by utilizing an antibody against a mental disorder-related protein Shati.

Many mentally ill disorders, such as schizophrenia and depression, develop early in life, such as adolescence, and patients must live with the disease for the rest of their lives along with drugs and behavioral therapy. There are restrictions on daily life and occupational choices. If treatment of such a disease can be started as early as possible, it can stop the worsening of the disease and find a possibility of recovery.
As a diagnosis of schizophrenia, for example, the amount of epithelial cell growth factor in serum is measured and judged by the reduced amount of the subject (Patent Document 1). There has been a report of determining by measuring the level of a protein involved in glutathione (Patent Document 2).

On the other hand, drug dependence due to stimulants and narcotics has become a major social problem, and functional molecules related to the formation of dependence have been searched with the aim of establishing therapeutic methods. As a result, Shati showing a high expression level in the nucleus accumbens of a stimulant psychosis model mouse
(NCBI access number ABA54615) was found (Non-Patent Document 1). Diagnosis of mental disorders by detecting Shati's nucleic acid has been proposed (Patent Document 3).

WO2002 / 037105 WO2005 / 068649 (Special Table 2007-524408) WO2006 / 093034

J. Neurosci., 27, 7604-7615,2007

The present invention provides a method capable of in vitro diagnosis of mental disorders such as drug dependence, schizophrenia, depression and the like, and a diagnostic kit for use in the diagnosis method, and a method for easily and early detecting mental disorders・ Providing tools is an issue.

The present inventor has studied behavioral pharmacological, biochemical, and cell biological aspects of Shati, focusing on its role in drug dependence formation and its effect on dopamine release. As a result, it was found that this protein has a deep relationship with mental illnesses such as schizophrenia and depression, and shows a high blood concentration in a psychotic model mouse administered with a stimulant. Furthermore, since a fragment derived from Shati is detected in urine, a kit for measuring the concentration of Shati or its fragment in blood or urine can be devised to diagnose early and simple mental illness causing psychiatric disorders. A method was established and the present invention was completed.
Hereinafter, the present invention will be described in detail.

The present invention collects blood of humans or animals (hereinafter referred to as “subjects”) and, as necessary, the amount of Shati itself or fragments derived from Shati in plasma or serum separated from blood by various methods. To quantify the amount of Shati-derived fragments in the subject's urine by various methods to diagnose the presence or condition of mental disorders resulting from drug addiction, schizophrenia, depression, etc. A diagnostic agent kit to be used and a method for diagnosing psychiatric disorders using the kit.

In the method of the present invention, the Shati or Shati fragment is preferably detected by sandwich ELISA (Enzyme-linked immunosorbent assay) with high Shati specificity.
The subject's blood or urine has a mental disorder due to drug dependence, schizophrenia, depression, etc., taking advantage of the fact that the concentration of Shati or Shati fragment in the blood or urine is higher than the concentration range of healthy individuals or healthy animals. Judge.

The present invention is a diagnostic kit for a psychiatric disorder characterized by comprising a solid phase, an antibody against Shati immobilized on the solid phase, and a labeled antiglobulin antibody.
In the present invention, the antiglobulin antibody is labeled with an enzyme label, a fluorescent label or a radiolabel.
Here, the enzyme label is a label with an enzyme such as peroxidase, β-D-galactosidase, alkaline phosphatase and glucose-6-phosphate dehydrogenase.
The diagnostic agent kit of the present invention also includes a detection reagent for detecting the label of the labeled globulin antibody, if necessary.

Furthermore, the present invention is a diagnostic kit for a psychiatric disorder comprising a solid phase, an antibody against Shati immobilized on the solid phase, a biotin-modified antiglobulin antibody, and labeled avidin that reacts with biotin.
Here, the labeled avidin is labeled with an enzyme such as peroxidase, β-D-galactosidase, alkaline phosphatase and glucose-6-phosphate dehydrogenase. If necessary, a detection reagent for detecting the label of the labeled avidin is also provided.

Furthermore, the present invention is a diagnostic kit for a mental disorder characterized by comprising a solid phase, an antibody against Shati, a biotin-modified antiglobulin antibody, and a labeled avidin that reacts with biotin.
Here, the labeled avidin is labeled with an enzyme such as peroxidase, β-D-galactosidase, alkaline phosphatase and glucose-6-phosphate dehydrogenase. If necessary, a detection reagent for detecting the label of the labeled avidin is also provided.

The meanings or definitions of terms used in this specification are described below.
“Shati” is a protein registered as ABA 54615 with the National Center for Biotechnology Information (NCBI). In addition, the “Shati fragment” means a peptide that is generated when a part of a peptide constituting Shati or Shati is decomposed in vivo.

“Antibody against Shati” refers to an antibody prepared using Shati as an antigen. The antibody only needs to have the ability to bind to Shati, and includes polyclonal antibodies, oligoclonal antibodies (mixtures of several to several tens of antibodies), and monoclonal antibodies. As a polyclonal antibody or oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. The anti-IgSF4 antibody may be an antibody fragment such as Fab, Fab ′, F (ab ′) 2, scFv, or dsFv antibody. Preferred examples include polyclonal antibodies and monoclonal antibodies that specifically bind to Shati.

The “labeled antiglobulin antibody” means that the antiglobulin antibody is labeled with an enzyme such as peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and labeled with fluorescence such as delphinium, Alternatively, it refers to an antibody devised so that an anti-globulin antibody can be quantified by labeling with a radioisotope. In the case of labeling with an enzyme, the bound anti-globulin antibody can be detected by reacting the enzyme with an appropriate substrate, using the enzyme reaction product as a marker. In the case of labeling with a fluorescent label or a radioisotope, the bound anti-globulin antibody can be detected using fluorescence or radioactivity as a marker.
Furthermore, the “labeled antiglobulin antibody” includes an antiglobulin antibody labeled with biotin, 2,4-dinitrophenol or the like. Biotin binds specifically to avidin and 2,4-dinitrophenol binds specifically to anti-2,4-dinitrophenol antibody. Therefore, avidin or anti-2,4-dinitrophenol antibody labeled with an enzyme such as peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase or the like is used. And can be quantified.

The diagnostic kit for mental disorders of the present invention is characterized by measuring the concentration of Shati or the concentration of a fragment derived from Shati in the blood, serum or urine of a subject using an anti-Shati antibody. The diagnostic kit for mental disorders of the present invention is useful for determining mental disorders resulting from drug addiction, schizophrenia, depression and the like. In addition, the diagnostic kit for mental disorders of the present invention is useful for knowing a pregnant woman's depression state and depression tendency.

It is a calibration curve in Shati's ELISA. Shati concentration in whole blood of stimulant psychotic mice and control mice. Shati concentration in serum of stimulant psychotic mice and control mice. Gel electrophoresis diagram of stimulant psychotic mouse and control mouse. Shati concentration in urine of stimulant psychotic mice and control mice. Shati concentration in maternal serum.

As a specific method for measuring the amount of Shati or the amount of fragments derived from Shati in blood, serum or urine, for example,
(1) a step of immobilizing an anti-Shati antibody on a solid phase such as polystyrene, nylon, glass, silicon rubber, sepharose;
(2) adding or contacting the subject's blood or urine to the solid phase;
(3) washing the solid phase;
(4) adding or contacting a labeled anti-globulin antibody;
(5) Step of measuring the amount of Shati or Shati fragment using the label (6) From the step of comparing the measured amount of Shati or Shati fragment with the range of measured values of healthy human or animal measured in advance The method which becomes.

In addition, as a specific method for measuring the amount of Shati or Shati fragment in blood or urine, for example,
(1) a step of immobilizing an anti-Shachi antibody on a solid phase such as polystyrene, nylon, glass, silicon rubber, sepharose;
(2) adding or contacting the subject's blood or urine to the solid phase;
(3) washing the solid phase;
(4) adding or contacting a biotin-modified antiglobulin antibody;
(5) adding or contacting labeled avidin;
(6) A step of measuring the amount of Shati or the amount of Shati fragment using the label;
(7) A method comprising a step of comparing the measured amount of Shati or Shati fragment with the range of measured values of a healthy human or animal measured in advance.

In addition, as a specific method for measuring the amount of Shati or Shati fragment in blood or urine, for example,
(1) A step of fixing a subject's blood or urine to a solid phase such as polystyrene, nylon, glass, silicon rubber, sepharose;
(2) adding or contacting the anti-Shachi antibody to the solid phase;
(3) washing the solid phase;
(4) adding or contacting a biotin-modified antiglobulin antibody;
(5) adding or contacting labeled avidin;
(6) A step of measuring the amount of Shati or the amount of Shati fragment using the label;
(7) A method comprising a step of comparing the measured amount of Shati or Shati fragment with the range of measured values of a healthy human or animal measured in advance.

Examples of the shape of the solid phase include small spheres, wells, test tubes, and the like. The use of a plate having wells as the solid phase is preferred. Further, in the case of a plate having a well as a solid phase, in the step of immobilizing the anti-Shati antibody, after the anti-Shati antibody is adsorbed to the well, the plate is placed at around 40 ° C. (40 ° C. ± 1 ° C.) for about 30 minutes (30 minutes (± 5 minutes) It is preferable to warm.

The antibody used in the present invention can be prepared using an immunological technique, a phage display method, a ribosome display method, or the like.
Preparation of a polyclonal antibody by an immunological technique can be performed, for example, by the following procedure.
An antigen (a protein of interest or a part thereof) is prepared and used to immunize animals such as rabbits, mice, rats, chickens, and turkeys. The antigen can be obtained by purifying a biological sample. A recombinant protein or peptide can also be used as an antigen.
For example, using a partial peptide (CNTAFRGLRQHPRTQLL, CMSVDSRFRGKGIAKALG) described in Patent Document 3 (WO2006 / 093034) as an antigen, an antibody having specific binding to Shati can be obtained.

The recombinant protein or peptide as an antigen is, for example, a gene encoding the amino acid sequence of SEQ ID NOs: 7 to 12 described in Patent Document 3 (which may be a part of the gene), E. coli, It can be prepared by introducing into a suitable host such as Bacillus subtilis, yeast, plant or animal cells and expressing in the resulting recombinant cells.
In order to enhance the immunity-inducing action, an antigen bound with a carrier protein may be used. Carrier proteins include KLM (Keyhole Light Hemocyanin) and BSA (Bovine Serum
Albumin), OVA (Ovalbumin), etc. can be used. A carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimidobenzoyloxysuccinimide) method, or the like can be used for carrier protein binding.
Alternatively, an antigen in which the target protein or a part thereof is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag, or the like can be used. Such a fusion protein can be easily purified by a general method.

Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

A monoclonal antibody can be prepared by the following procedure.
First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone.

On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. The culture medium or the ascites may be purified by affinity chromatography using protein G, protein A or the like. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods may be used alone or in any combination.

Psychiatric disorders can be diagnosed using the diagnostic kit of the present invention. That is, by determining the amount of Shati in blood or animal urine collected from humans or animals by various methods, it is determined whether the amount of Shati exceeds the range of healthy humans or animals. The presence or absence can be diagnosed.
As shown in the Examples below, in drug-dependent model mice, Shati in the blood is present at a higher concentration than in control normal mice.
Therefore, by using the diagnostic kit of the present invention, by measuring the Shati content of blood or urine of an animal such as a human or mouse as a subject, it is possible to know whether or not there is a mental disorder and the degree of drug dependence. It is possible to know whether or not a person suffers from a psychiatric disorder such as symptom, schizophrenia, depression, and the progress of the disease. Specifically, for example, by using the blood or urine of a pregnant woman as a specimen and using the diagnostic agent kit of the present invention, the depression tendency of the pregnant woman can be known.

Hereinafter, although a reference example and an example explain the present invention, the present invention is not limited by these.
Reference example 1
(1) Shati protein
COS cells were transformed by incorporating killer whale cDNA into pDEST. Culture was performed according to a conventional method, and the cells were physically detached in a phosphate buffer.

(2) A peptide based on the sequence of shati (“CNTAFRGLRRQHPRTQLL” described in Patent Document 3) was subcutaneously administered to a Shati antibody rabbit (New Zealand, White). Seven doses were given at one week intervals, and whole blood was collected at week 7 to separate the serum. Protein A was used to purify IgG in serum.

(3) Stimulant-induced psychosis model mouse
Methamphetamine (10 mg / kg) was administered once a day to 6-week-old male ICR mice (Japan SLC). Urine was collected after 10 days of administration. Blood at the time of decapitation was collected to obtain serum components. These biological components were used as specimens for enzyme immunoassay. For urine, a sample was prepared by adding the same volume of 0.1 M Tris / HCl buffer (pH 7.4) containing 1% bovine serum albumin and 0.2 M sodium chloride.

(4) Preparation of standard solution
Shati protein was diluted from 0.1 pg to 1 μg / mL with 0.1 M Tris / HCl buffer (pH 8.5).

Example 1
Shati antibody (1 mg / mL) was added dropwise to a 96-well plate at 5 μL per well. The plate was covered with parafilm to prevent evaporation of the diluent. The 96-well plate was placed on a 40 ° C. hot plate for 30 minutes. Then, it incubated at 4 degreeC overnight. After removing the antibody solution by suction, wash it with 150 μL of washing buffer (0.1 M Tris / HCl, 0.1% bovine serum albumin, 0.2 M sodium chloride, 100 mM magnesium chloride, 0.2% sodium azide, pH 7.4) three times. did. 30 μL of the sample or standard solution was added and allowed to react for 5 hours at room temperature under shaking. The reaction solution was removed by suction, and then washed 3 times with a washing buffer. 30 μL of biotinylated anti-rabbit IgG bovine antibody (1 mg / mL) was allowed to react at 4 ° C. overnight. After aspirating and removing the biotinylated anti-rabbit IgG bovine antibody, 30 μL of β-D-galactidase-labeled streptavidin (diluted 0.1 μg / mL with a washing buffer solution) was added and reacted at room temperature for 1 hour. 4-Methylumbelliferone-β-D-galactoviranoside (diluted to 10 μg / mL with a washing buffer) was added as a substrate, and the reaction was allowed to proceed at room temperature for 5 hours under light shielding and shaking. The reaction was stopped by adding 100 μL of 0.1 M glycine-sodium hydroxide buffer (pH 10.3), and the fluorescence intensity of the produced 4-methylumbelliferone was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm.

Example 2
Samples or standard solutions were placed in 96-well plates in 30 μL aliquots and incubated overnight at 4 ° C. After removing the protein solution by suction, it is washed 3 times with 150 μL of washing buffer (0.1 M Tris-HCl, 0.1% bovine serum albumin, 0.2 M sodium chloride, 100 mM magnesium chloride, 0.2% sodium azide, pH 7.4) did. Blocking was performed for 1 hour with a 1% skim milk solution diluted with 150 μL of washing buffer.
After removing the 1% skim milk solution by suction, 30 μL of anti-Shati antibody (1 mg / mL) dissolved in a washing buffer solution was added and left at 4 ° C. overnight.
30 μL of biotinylated anti-rabbit IgG bovine antibody (1 μg / mL) is reacted for 5 hours. After aspiration removal, wash 3 times with 150 μL washing buffer, add 30 μL β-D-galactidase-labeled streptavidin (diluted to 0.1 μg / mL with washing buffer), and 1 hour at room temperature Reacted. 4-Methylumbelliferone-β-D-galactoviranoside (diluted to 10 μg / mL with a washing buffer) was added as a substrate, and the reaction was allowed to proceed at room temperature for 5 hours under light shielding and shaking. The reaction was stopped by adding 100 μL of 0.1 M glycine-sodium hydroxide buffer (pH 10.3), and the fluorescence intensity of the produced 4-methylumbelliferone was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm.

Example 3
Under the approval of the University of Toyama Ethics Committee, blood was collected from patients who gave explanations and consented in writing. Serum components were removed from collected human blood by centrifugation, and added with 19 volumes of homogenizing buffer (0.1 M Tris buffer containing 1 M sodium chloride, 2% bovine serum albumin, 2 mM EDTA) as a sample, Measurements were performed according to the method described in Example 2.

With the diagnostic kit of the present invention, by measuring the Shati content of blood or urine of an animal such as a human being or a subject, it is possible to know whether or not there is a mental disorder, and the subject is drug dependent, You can know whether you are suffering from a psychiatric disorder such as schizophrenia or depression, and the progress of the disease. Therefore, it is useful for the treatment of mental disorders such as drug addiction, schizophrenia and depression.

Claims (15)

A method for diagnosing a mental disorder to determine whether or not a subject's nerve is impaired,
(1) The process of collecting blood from a subject or collecting urine from a subject,
(2) a process of measuring the concentration of Shati in the blood or urine using an antibody against Shati, and (3) when the concentration of Shati in the subject exceeds the concentration range of a healthy subject, A method for diagnosing a mental disorder comprising the process of determining that the subject has a mental disorder. The method for diagnosing a mental disorder according to claim 1, wherein the mental disorder is caused by any one of drug dependence, schizophrenia, and depression. The method for diagnosing a mental disorder according to claim 2, wherein the depression is depression or a tendency of depression in a pregnant woman. A diagnostic kit for mental disorders, comprising a solid phase, an antibody against Shati immobilized on the solid phase, and a labeled antiglobulin antibody. The diagnostic kit for mental disorders according to claim 4, wherein the mental disorder is caused by any one of drug dependence, schizophrenia, and depression. 6. The method for diagnosing a mental disorder according to claim 5, wherein the depression is maternal depression. The diagnostic kit for mental disorders according to claim 4, wherein blood or urine is used as a specimen. A diagnostic kit for mental disorders, comprising a solid phase, an antibody against Shati immobilized on the solid phase, a biotin-labeled antiglobulin antibody, and an enzyme-labeled avidin. 9. The diagnostic kit for mental disorders according to claim 8, wherein the mental disorder is caused by any one of drug dependence, schizophrenia, and depression. The method for diagnosing a mental disorder according to claim 9, wherein the depression is a maternal depression. The diagnostic kit for psychiatric disorders according to claims 8 to 10, wherein blood or urine is used as a specimen. A diagnostic kit for mental disorders, comprising a solid phase, an antibody against Shati, a biotin-labeled antiglobulin antibody, and an enzyme-labeled avidin. The diagnostic kit for a mental disorder according to claim 12, wherein the mental disorder is caused by any one of drug dependence, schizophrenia, and depression. The method for diagnosing a mental disorder according to claim 13, wherein the depression is maternal depression. 15. The diagnostic kit for mental disorders according to claim 12, wherein blood or urine is used as a specimen.