JP4834835B2 - Diagnostic agent for autism - Google Patents

Description

  The present invention relates to a diagnostic agent for autism. More specifically, the present invention relates to an invention of a diagnostic agent for autism mainly comprising an antibody against a biological protein newly found to be effective as an index for diagnosis of autism, and an invention related thereto. .

  Autism is a disorder first described as “Autistic Disorder of Emotional Contact” in 1943 by Dr. Canner, USA. The onset is very early in life, and the main symptoms are extreme isolation and autism from the surroundings, unusual language symptoms, and compulsive identity retention. Autism is a developmental disorder in which a disorder appears early in life and changes its state depending on the developmental process. It is presumed to be a disorder of the higher central nervous system that has not yet been identified.

  High-functioning autism, on the other hand, appears by about the age of three, and is characterized by difficulty in forming social relationships with others, slow language development, and narrow interest and interest in specific things. Autism that is a behavioral disorder that does not involve a delay in intellectual development. The prevalence of autism is 7 to 16 per 10,000 children, and high-functioning autism is reported to be approximately 11 to 34% of autism (Non-patent Document 1). The cause of high-functioning autism is not clear at the present time, but biological factors based on brain dysfunction and developmental disorders are intricately intertwined with psychological factors from the environment during development It is speculated.

Macintosh, KE et al., Annotation: The similarities and differences between autistic disorder and Asperger's disorder: a review of the empirical evidence.Journal of Child Psychology and Psychiatry. (2004) 45: 421-434. Interviews using the Medical Society Diagnostic Standards (DSM-IV), World Health Organization WHO ICD-10, and ADI-R (Autism Diagnostic Interview-Revised). In autism, enlargement of the head circumference is observed early after birth, and it is considered that there is an increase in the formation of neurons and glia or a decrease in cell death during this period. Furthermore, it is strongly suggested that nerve growth factors are involved in this process (Non-Patent Document 2). In addition, due to the peculiarities of the pathology of autism, establishment of a comprehensive treatment system such as early diagnosis, early treatment, rehabilitation activities, and prevention is desired, but the biological marker of autism is Not yet established.

McCaffary, P et al., Macrocephaly and the control of brain growth in autistic disorders. Progress in Neurobioligy (2005) 77: 38-56. By the way, Transforming Growth Factor beta 1 (hereinafter referred to as “TGFβ1”) Is one of the multifunctional regulatory cytokine subtypes involved in development and tissue repair. TGFβ is produced in glia and nerve cells in the brain, and TGFβ1 is thought to be a cytokine mainly related to injury repair, but also plays an important role as a regulatory factor in the nervous system (Non-patent Document 3). ). TGFβ1 and its receptor are expressed in the developing nervous system, suggesting that they are involved in brain development (Non-patent Document 4) and are accompanied by extensive cell death in TGFβ1 knockout mice. Serious cortical developmental disorder is observed (Non-patent Document 5). From these, considering the role of TGFβ1 in brain development, it may be related to the pathophysiology of autistic patients.

Gomez, FC et al., Emerging roles for TGF-betal in nervous system development. Int J Dev Neurosci (2005) 23: 413-424. Bautner, M et al., The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions. J Neurochem (2000) 75: 2227-2240. Brionne, TC et al., Loss of TGF-betal leads to increased neuronal cell death and microgliosis in mouse brain. Neuron (2003) 40: 1133-1145. Next, hepatocyte growth factor (HGF) Is present not only in the liver but also in the kidney, lungs, cardiovascular system and nervous system, and plays a role in organ regeneration and protection. In addition, HGF is an embryonic neuron in rodents, such as promoting interneuron migration from the ventral side to the dorsal side of the telencephalon (Non-patent Document 6) and controlling the proliferation and differentiation of granule cells (Non-patent Document 7). It is involved in development (Non-patent Document 8). Furthermore, it has been found that HGF regulates the formation of cerebral cortical pyramidal dendrites in the neonatal period and is essential for the development of nerve cells (Non-patent Document 9). Considering the role of HGF in these brain developments, it is considered that HGF is involved in the pathophysiology of autistic patients (Non-patent Document 10). In recent years, it has been reported that HGF in cerebrospinal fluid of autistic patients is significantly higher than that in the healthy group (Non-patent Document 11), and abnormalities in HGF concentration in peripheral blood are estimated. There are no reports so far.

Powell, EM et al., Hepatocyte growth factor / scatter factor is a motogen for interneuronsmigrating from the ventral to dorsal telencephalon.Neuron (2001) 30: 79-89. Aeraki, A et al., Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development.Proc Natl Acad Sci USA (2002) 99: 15200-15205. Mina, F et al., Hepatocyte growth factor, a versatile signal for developing neurons. Nature Neurosci (1999) 2: 213-217. Gutierrez, H et al., HGF regulates the development of cortical pyramidaldendrites. Development (2004) 131: 3717-3726. Levit, P et al., Regulation of neocortical interneuron development and the implications for neurodevelopmental disorders. Trends Neurosci (2004) 27: 400-406. Vargas, DL et al., Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol (2005) 57: 67-81. In addition, epidermal growth factor (hereinafter referred to as “EGF”) and related factors are , It binds to the EGF receptor and promotes the proliferation of fibroblasts and epithelial cells, and plays an important role in the suppression of proliferation, differentiation, angiogenesis, and apoptosis. In addition, EGF has been confirmed to be present in neurons and astrocytes in adult brain, and considering the role of EGF in cranial nerves, it is considered that EGF is involved in the pathophysiology of autistic patients (non-patent literature). 12). Furthermore, since radiolabeled EGF administered into blood rapidly passes through the cerebral blood flow barrier, it is considered that EGF concentration in peripheral blood reflects EGF concentration in the brain (Non-patent Document 13). .

Ferrer, I et al., Transforming growth factor-alpha (TGF-) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain.Prog Neurobiol (1996) 49: 99-123. Pan, W et al., Entry of EGF into brain is rapid and saturable. Peptides (1999) 20: 1091-1098.

  As described above, a simple biological marker has not been established for autism, it is difficult to diagnose, and appropriate measures cannot be taken early, which further worsens the symptoms. It is a cause. Therefore, development of diagnostic agents and diagnostic methods capable of early diagnosis of autism is desired from the medical field.

  As a result of intensive studies to solve the above problems, the present inventors have found that the levels of TGFβ1, HGF, and EGF in the serum of autistic patients are significantly lower than those of healthy individuals. I found out.

  Then, by utilizing such differences in the levels of TGFβ1, HGF or EGF, anti-transforming growth factor beta 1 antibody (anti-TGFβ1 antibody), anti-hepatocyte growth factor antibody (anti-HGF antibody) or anti-epidermal growth factor It was found that an antibody (anti-EGF antibody) can be used for early, simple and reliable diagnosis of autism. The present invention has been completed based on such findings.

[Meaning and definition of terms]
Hereinafter, the meaning or definition of terms in this specification will be described.

  “Autism” refers to extreme isolation and autism from the surroundings as a major symptom, peculiar language symptoms, and compulsive identity retention. It is a developmental disorder whose state changes with the process. “High-functioning autism” is manifested by about 3 years old and is characterized by difficulty in forming social relationships with others, delay in language development, narrow interest and interest in specific things Autism that is a behavioral disorder that does not involve a delay in intellectual development.

  “Blood sample” means blood collected from humans (not limited to autistic patients) or animals (for example, autism model animals), as well as serum prepared from these blood, composition containing blood or serum ( For example, it is a concept including, without limitation, dilution with a pH buffer solution), or lyophilized products thereof.

  “Anti-transforming growth factor beta 1 antibody” refers to an antibody purified using transforming growth factor beta 1 as an antigen. The antibody only needs to have the ability to bind to transforming growth factor beta1, and includes polyclonal antibodies and monoclonal antibodies. Preferable examples include polyclonal antibodies and monoclonal antibodies that specifically bind to transforming growth factor beta1.

  “Labeled anti-transforming growth factor beta 1 antibody” refers to transforming growth factor beta 1 antibody as peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydration. An antibody devised so that transforming growth factor beta 1 can be quantified by binding an enzyme such as elementary enzyme, a fluorescent label such as delphinium, a radioisotope label or isotope label, biotin and the like.

  Furthermore, “labeled anti-transforming growth factor beta 1 antibody” also includes anti-transforming growth factor beta 1 modified with biotin, 2,4-dinitrophenol and the like. In this case, it can be quantified by further using avidin labeled with the labeled anti-transforming growth factor beta 1 antibody and labeled anti-2,4-dinitrophenol antibody.

  “Anti-hepatocyte growth factor antibody” refers to an antibody purified using hepatocyte growth factor as an antigen. The antibody only needs to have the ability to bind to hepatocyte growth factor, and includes polyclonal antibodies and monoclonal antibodies. Preferable examples include polyclonal antibodies and monoclonal antibodies that specifically bind to hepatocyte growth factor.

  “Labeled anti-hepatocyte growth factor antibody” refers to hepatocyte growth factor antibody such as peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, etc. An antibody devised so that hepatocyte growth factor can be quantified by binding an enzyme, a fluorescent label such as delphinium, a radioisotope label or isotope label, biotin and the like.

  Furthermore, “labeled anti-hepatocyte growth factor antibody” also includes anti-hepatocyte growth factor modified with biotin, 2,4-dinitrophenol and the like. In this case, the labeled anti-hepatocyte growth factor antibody can be quantified by further using avidin and labeled anti-2,4-dinitrophenol antibody.

  “Anti-epidermal growth factor antibody” refers to an antibody purified using epidermal growth factor as an antigen. The antibody only needs to be capable of binding to epidermal growth factor and includes polyclonal antibodies and monoclonal antibodies. Preferable examples include polyclonal antibodies and monoclonal antibodies that specifically bind to epidermal growth factor.

  "Labeled anti-epidermal growth factor antibody" refers to an epidermal growth factor antibody such as peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, An antibody devised to bind to a fluorescent label such as delphinium, a radioisotope label or isotope label, biotin, etc., and to quantify epidermal growth factor.

  Furthermore, the “labeled anti-epidermal growth factor antibody” also includes an anti-epidermal growth factor modified with biotin, 2,4-dinitrophenol or the like. In this case, the labeled anti-epidermal growth factor antibody can be further quantified by using avidin and labeled anti-2,4-dinitrophenol antibody.

  Note that the above TGFβ1, HGF, EGF, anti-TGFβ1 antibody, anti-HGF antibody, and anti-EGF antibody are all known per se, and therefore the description of these amino acid sequences and the like is omitted in this specification.

[Invention related to TGFβ1]
The invention groups related to TGFβ1 among the present invention are as follows.

  1) A diagnostic agent for autism mainly comprising an anti-TGFβ1 antibody.

  2) The diagnostic agent for autism according to 1) above, which is used for measuring the TGFβ1 concentration in a human blood sample.

  3) The diagnostic agent for autism according to 1) or 2) above, comprising an unlabeled anti-TGFβ1 antibody and / or a labeled anti-TGFβ1 antibody.

  4) An autism diagnostic kit comprising the diagnostic agent for autism according to any one of 1) to 3) above.

  5) The autism diagnostic kit according to 4), which is used for measuring the TGFβ1 concentration in a human blood sample.

  6) Autism test for measuring TGFβ1 concentration in a human blood sample using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above Method.

  7) An assay method for examining the effect of a therapeutic agent for autism using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above.

  8) In a blood sample of an animal including at least an autism model animal using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above A method for assaying autism in which TGFβ1 concentration is measured.

  9) A therapeutic agent for autism, which is a compound that increases TGFβ1 in the human body.

[Invention related to HGF]
Among the present invention, the invention groups related to HGF are as follows.

  1) A diagnostic agent for autism mainly composed of an anti-HGF antibody.

  2) The diagnostic agent for autism according to 1) above, which is used for measuring the HGF concentration in a human blood sample.

  3) The diagnostic agent for autism according to 1) or 2) above, comprising an unlabeled anti-HGF antibody and / or a labeled anti-HGF antibody.

  4) An autism diagnostic kit comprising the diagnostic agent for autism according to any one of 1) to 3) above.

  5) The autism diagnostic kit according to 4), which is used for measuring HGF in a human blood sample.

  6) Autism test for measuring the HGF concentration in a human blood sample using the diagnostic agent for autism according to 1) to 3) above or the autism diagnostic kit according to 4) or 5) above Method.

  7) An assay method for examining the effect of a therapeutic agent for autism using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above.

  8) In a blood sample of an animal including at least an autism model animal using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above A method for assaying autism in which the HGF concentration is measured.

  9) A therapeutic agent for autism, which is a compound that increases HGF in the human body.

[Invention related to EGF]
Among the present inventions, the invention groups related to EGF are as follows.

  1) A diagnostic agent for autism mainly composed of an anti-EGF antibody.

  2) The diagnostic agent for autism according to 1) above, which is used for measuring EGF concentration in a human blood sample.

  3) The diagnostic agent for autism according to 1) or 2) above, comprising an unlabeled anti-EGF antibody and / or a labeled anti-EGF antibody.

  4) An autism diagnostic kit comprising the diagnostic agent for autism according to any one of 1) to 3) above.

  5) The autism diagnostic kit according to 4), which is used for measuring EGF in a human blood sample.

  6) Autism test for measuring EGF concentration in a human blood sample using the autism diagnostic agent of any of 1) to 3) above or the autism diagnostic kit of 4) or 5) above Method.

  7) An assay method for examining the effect of a therapeutic agent for autism using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above.

  8) In a blood sample of an animal including at least an autism model animal using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above A method for testing autism in which EGF concentration is measured.

  9) A therapeutic agent for autism, which is a compound that increases EGF in the human body.

[Inventions related to multiple factor systems]
Among the present invention, the invention groups related to the multi-factor system targeting two or more selected from TGFβ1, HGF, and EGF are as follows.

  1) A diagnostic agent for autism mainly comprising any two or more of anti-TGFβ1 antibody, anti-HGF antibody and anti-EGF antibody.

  2) The diagnostic agent for autism according to 1), which is used for measuring any two or more of TGFβ1 concentration, HGF concentration and EGF concentration in a human blood sample.

  3) The diagnostic agent for autism according to 1) or 2) above, wherein the anti-TGFβ1 antibody, anti-HGF antibody, and anti-EGF antibody are respectively unlabeled and / or labeled.

  4) An autism diagnostic kit comprising the diagnostic agent for autism according to any one of 1) to 3) above. Any kit for autism diagnosis that includes at least one of the above-described autism diagnostic agents corresponds to this autism diagnosis kit. The autism diagnostic kit can include other optional components that may be included in this type of kit, for example, components such as a buffer for dilution.

  5) The autism diagnostic kit according to 4), which is used for measuring any two or more of TGFβ1, HGF and EGF concentrations in a human blood sample.

  6) Using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above, the TGFβ1 concentration, HGF concentration and EGF concentration in a human blood sample Autism test method to measure any two or more of the above.

  7) An assay method for examining the effect of a therapeutic agent for autism using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above.

  8) In a blood sample of an animal including at least an autism model animal using the autism diagnostic agent of any one of 1) to 3) above or the autism diagnostic kit of 4) or 5) above A method for assaying autism in which any two or more of TGFβ1 concentration, HGF concentration and EGF concentration are measured.

  The above means enables early, simple and reliable diagnosis of autism. In particular, the concentrations of TGFβ1, HGF, and EGF in a human (patient) blood sample are measured using an anti-TGFβ1 antibody, an anti-HGF antibody, an anti-EGF antibody, or a labeled antibody obtained by labeling them with an appropriate means. Thus, diagnosis of autism can be easily performed.

  Further, when using an autism diagnostic agent mainly comprising any two or more of these antibodies or an autism diagnostic kit comprising this autism diagnostic agent, two or more types are used. By simultaneously performing an autism diagnosis based on the above index, the reliability of the diagnosis can be further enhanced.

  Next, modes for carrying out the present invention will be described including the best mode.

[Embodiments of the Invention Related to TGFβ1, HGF or EGF]
The diagnosis of autism according to the present invention can be performed, for example, as follows. Serum is prepared from human blood and the amount of TGFβ1, HGF or EGF in the serum is quantified by various methods. Desirably, TGFβ1, HGF or EGF is detected and quantified by sandwich ELISA using an antibody having high specificity for TGFβ1, HGF or EGF. Autism can be diagnosed by utilizing the fact that the concentration of TGFβ1, HGF, or EGF in the serum of autistic patients is significantly lower than that in healthy individuals.

  As a specific method for measuring TGFβ1, HGF or EGF in serum, for example, a method comprising the following steps can be mentioned.

1. Immobilizing an anti-TGFβ1 antibody, an anti-HGF antibody or an anti-EGF antibody on a solid phase such as polystyrene, nylon, glass, silicon rubber, or cephalose;
2. Adding or contacting the serum of a patient to be diagnosed to a solid phase,
3. Washing the solid phase,
4). Adding or contacting a labeled anti-TGFβ1 antibody, anti-HGF antibody or anti-EGF antibody,
5). A step of measuring the amount of TGFβ1, HGF or EGF using the label.

  Furthermore, as a specific method for measuring TGFβ1, HGF or EGF in serum, for example, a method comprising the following steps can be mentioned.

1. Immobilizing an anti-TGFβ1 antibody, an anti-HGF antibody or an anti-EGF antibody on a solid phase such as polystyrene, nylon, glass, silicon rubber, or cephalose;
2. Adding or contacting the serum of a patient to be diagnosed to a solid phase,
3. Washing the solid phase,
4). Adding or contacting anti-TGFβ1 antibody, anti-HGF antibody or anti-EGF antibody modified with biotin or 2,4-dinitrophenol,
5). Adding or contacting labeled avidin or labeled 2,4-dinitrophenol antibody;
6). A step of measuring the amount of TGFβ1, HGF or EGF using the label.

  Furthermore, as a specific method for measuring TGFβ1, HGF or EGF in serum, for example, a method comprising the following steps can be mentioned. In this method, the shape of the solid phase includes small spheres, wells, test tubes and the like.

1. Immobilizing an anti-TGFβ1 antibody, an anti-HGF antibody or an anti-EGF antibody on a solid phase such as polystyrene, nylon, glass, silicon rubber, or cephalose;
2. Adding or contacting the blood of the patient to be diagnosed to the solid phase,
3. Washing the solid phase,
4). Adding or contacting an anti-TGFβ1 antibody, anti-HGF antibody or anti-EGF antibody modified with biotin;
5). Adding or contacting labeled avidin;
6). A step of measuring the amount of TGFβ1, HGF or EGF using the label.

  Since TGFβ1, HGF, or EGF used as an antigen or ELISA standard is commercially available, they can be used or prepared by the following method.

  When genetic engineering techniques are used, a gene encoding TGFβ1, HGF or EGF is inserted into an appropriate vector, inserted into an appropriate host, transformed, and the target set is obtained from the culture supernatant of this transformation. Recombinant TGFβ1, recombinant HGF, or recombinant EGF can be obtained, respectively, and is suitable for the production of homogeneous large amounts of TGFβ1, HGF, or EGF. The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as E. coli, Bacillus subtilis, yeast, plant or animal cells can be used.

  Anti-TGFβ1 antibody, anti-HGF antibody or anti-EGF antibody is prepared by immunizing rabbits, chickens, turkeys and the like using TGFβ1, HGF or EGF as an antigen, respectively. For the labeled anti-TGFβ1 antibody, labeled anti-HGF antibody, or labeled anti-EGF antibody, use a commercially available kit of a biotinylation reagent or a peroxidase with a cross-linking agent for the anti-TGFβ1 antibody, anti-HGF antibody, or anti-EGF antibody, respectively. Can be reacted and prepared.

  This method is also useful for determining a therapeutic agent for autism. That is, a compound having an action of increasing TGFβ1, HGF, or EGF in the human body or model animal body may be useful as a therapeutic agent for autism. A model animal (such as a mouse or rat) having a low amount of TGFβ1, HGF or EGF is also useful as an animal model of autism. Therefore, by using this assay method, it is possible to screen for a new therapeutic agent for autism.

  The therapeutic agents found in such a manner can include drugs that can be administered parenterally or orally. The exact dosage and dosage regimen of the therapeutic agent will need to depend on the required amount for each individual subject, the method of treatment, the degree of the disease or need, and of course the judgment of the physician.

  The dosage and frequency of administration are not limited when administered parenterally, but vary depending on symptoms, age, body weight, dosage form, etc. For example, when administered subcutaneously or intravenously as an injection, 1 kg body weight of an adult patient per day The dose is selected from the range of about 0.1 mg to about 2500 mg, preferably about 1 mg to about 500 mg. For example, when administered to the trachea as a spray, the weight of an adult patient is 1 kg, about 0.1 mg to about The dosage is selected from the range of about 2500 mg, preferably from about 1 mg to about 500 mg. The administration schedule includes daily administration, intermittent administration, or a combination thereof.

  The dose and frequency of administration when administered orally are not limited, but vary depending on symptoms, age, body weight, dosage form, etc., for example, 1 kg body weight of an adult patient, a range of about 0.5 mg to about 2500 mg per day, Preferably, the dose is selected from the range of about 1 mg to about 1000 mg.

  A pharmaceutical composition can be produced by mixing the therapeutic agent for autism obtained by this method with a pharmaceutically acceptable non-toxic carrier. When preparing such compositions for parenteral administration (subcutaneous injection, intramuscular injection or intravenous injection), solution or suspension dosage forms are particularly good, especially for vaginal or rectal administration. Semi-solid dosage forms such as creams or suppositories are preferred, and for nasal administration, powders, nasal drops, or aerosol dosage forms are particularly preferred.

  The composition can be administered in a single dosage form and is well known in the pharmaceutical art as described, for example, in Remington's Pharmaceutical Sciences (Mac Publishing Company, Easton, PA, 1970). It can be prepared by any known method.

  Injectable preparations can contain, for example, plasma-derived proteins such as albumin, amino acids such as glycine, and sugars such as mannitol as pharmaceutical carriers. When used in an injection form, a buffer, a solubilizing agent, an isotonic agent and the like can be further added. In addition, when used as a water-soluble preparation or lyophilized preparation, it is preferable to add a surfactant such as Tween 80 (registered trademark) or Tween 20 (registered trademark) in order to prevent aggregation.

  Also, parenteral dosage forms other than injections may contain distilled water or physiological saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, hydrogenated naphthalene and the like. For example, preparations for vaginal or rectal administration, such as suppositories, contain, for example, polyalkylene glycol, petrolatum, cacao oil and the like as common excipients. Vaginal preparations may contain absorption enhancers such as bile salts, ethylenediamine salts, and citrates. Inhalation formulations may be solid, may contain, for example, lactose as an excipient, and nasal drops may be water or oil solutions.

[Embodiments of Invention Related to Multiple Factor System]
Among the present inventions, the invention relating to a multifactor system targeting two or more selected from TGFβ1, HGF, and EGF also relates to (1) its diagnosis, and (2) a specific method for measuring TGFβ1, HGF, or EGF. (3) Preparation of TGFβ1, HGF or EGF to be used as a standard for antigen or ELISA, (4) Preparation of anti-TGFβ1, anti-HGF or anti-EGF antibody, (5) Screening for a drug for treating autism -The above-mentioned various embodiments relating to utilization for ning, (6) dosage and schedule of therapeutic agent, (7) preparation as a pharmaceutical composition, and the like can be similarly performed.

  Next, examples of the present invention will be described. The technical scope of the present invention is not limited by these examples.

[Example 1: TGFβ1]
(1) Subjects 19 male patients with high-functioning autism [mean age: 23.4 years (standard deviation 2.6), age range: 18 to 28 years] were selected as subjects, and the health of the same age group 21 persons (average age: 22.7 years old (standard deviation 2.3), age range: 18 to 26 years old) were also selected as subjects as normal controls. All patients were diagnosed according to ADI-R (Autism Diagnostic Interview-Revised), a diagnostic criteria for high functioning autism.
(2) Experimental method A serum sample of a subject was collected and stored at -80 ° C until measurement. Serum levels of TGFβ1 were measured using a transforming growth factor beta 1 measurement kit (R & D Systems, USA) according to the manufacturer's instructions.

That is, using a 96-well plate coated with a synthetic transforming growth factor beta 1 type II receptor, 200 μL of diluted serum was added. As a standard for quantification, synthetic human TGFβ1 (31.2-2000 pg / mL) was added. After reacting at room temperature for 3 hours, it was washed 3 times with a buffer solution. Thereafter, an anti-horseradish peroxidase-labeled TGFβ1-specific polyclonal antibody (200 μL) was added and allowed to react at room temperature for 90 minutes. Next, after washing 3 times with a buffer solution, a TMB solution (200 μL) is added, and the mixture is allowed to react for 20 minutes at room temperature, protected from light. The absorbance at wavelengths of 450 nm and 550 nm was measured with an automatic microplate reader (EL340I, BIO-TEC, USA). The content of TGFβ1 in the specimen was measured by sandwich ELISA, and the concentration of TGFβ1 was calculated from the calibration curve.
(3) Statistical analysis Data are shown as mean ± standard deviation. Statistical analysis between the two groups was analyzed with Mann-Whitney U-test. A p value of 0.05 or less was considered statistically significant.
(4) Results i) Concentration of serum TGFβ1 in all subjects FIG. 1 shows the sprayed state of serum TGFβ1 concentration in normal controls and subjects with high-functioning autism.

  As seen in the above experimental results, the serum TGFβ1 concentration (average value: 7.34 ng / ml) of patients with high-functioning autism is the concentration of normal controls in the same age group (average value: 14.48 ng / ml). It was found to be significantly lower than Since TGFβ1 plays an extremely important role in the developmental process of the brain, the decreased serum TGFβ1 value is thought to contribute to the pathophysiology of high-functioning autism. For example, when the patient's serum TGFβ1 value is lower than the average value of the serum TGFβ1 value of the healthy subject minus the standard deviation value (12.84 pg / ml), the patient is diagnosed with high-functioning autism.

  In short, this experiment confirmed that TGFβ1 plays an extremely important role in the pathophysiology of autism, and that blood TGFβ1 is useful as a biological marker for autism.

[Example 2: HGF]
(1) Subjects 19 male patients with high-functioning autism [mean age: 23.4 years (standard deviation 2.6), age range: 18 to 28 years] were selected as subjects, and the health of the same age group 21 persons (average age: 22.7 years old (standard deviation 2.3), age range: 18 to 26 years old) were also selected as subjects as normal controls. All patients were diagnosed according to ADI-R (Autism Diagnostic Interview-Revised), a diagnostic criteria for high functioning autism.
(2) Experimental method A serum sample of a subject was collected and stored at -80 ° C until measurement. The serum level of HGF was measured using a hepatocyte growth factor measurement kit (R & D Systems, USA) according to the manufacturer's instructions.

That is, anti-hepatocyte growth factor monoclonal antibody was coated on a 96-well plate, and a buffer solution (150 μL) and 50 μL of diluted serum were added. A standard to which human HGF (125-8000 pg / mL) was added was used as a standard for quantification. After reacting at room temperature for 2 hours, it was washed 3 times with a buffer solution. Thereafter, an anti-horseradish peroxidase-labeled HGF polyclonal antibody (200 μL) was added and reacted at room temperature for 2 hours. Next, after washing 3 times with a buffer solution, a TMB solution (200 μL) is added, and after light-shielding and reacting at room temperature for 30 minutes, a stop solution (2N sulfuric acid: 50 μL) is added to stop the reaction, and 30 minutes The absorbance at wavelengths of 450 nm and 550 nm was measured with an automatic microplate reader (EL340I, BIO-TEC, USA). The content of HGF in the sample was measured by a sandwich type ELISA method, and the concentration of HGF was calculated from a calibration curve.
(3) Statistical analysis Data are shown as mean ± standard deviation. Statistical analysis between the two groups was analyzed with Mann-Whitney U-test. A p value of 0.05 or less was considered statistically significant.
(4) Results i) Concentration of serum HGF in all subjects FIG. 2 shows the distribution of serum HGF concentration in normal controls and subjects with high-functioning autism.

  As seen in the above experimental results, the serum HGF concentration (mean value: 505.2 pg / ml) of patients with high-functioning autism is the concentration of normal controls in the same age group (mean value: 825.2 ng / ml) It was found to be significantly lower than Since HGF plays an extremely important role in the developmental process of the brain, the decreased serum HGF level is thought to contribute to the pathophysiology of high-functioning autism. For example, when the patient's serum HGF level is lower than the average value of the serum HGF level of healthy subjects minus the standard deviation value (607.0 pg / ml), a diagnosis of high functioning autism is made.

  In short, this experiment confirmed that HGF plays a very important role in the pathophysiology of autism, and blood HGF is useful as a biological marker for autism.

[Example 3: EGF]
(1) Subjects 19 male patients with high-functioning autism [mean age: 23.4 years (standard deviation 2.6), age range: 18 to 28 years] were selected as subjects, and the health of the same age group 21 persons (average age: 22.7 years old (standard deviation 2.3), age range: 18 to 26 years old) were also selected as subjects as normal controls. All patients were diagnosed according to ADI-R (Autism Diagnostic Interview-Revised), a diagnostic criteria for high functioning autism.
(2) Experimental method A serum sample of a subject was collected and stored at -80 ° C until measurement. The serum level of EGF was measured using an epidermal growth factor measurement kit (R & D Systems, USA) according to the manufacturer's instructions.

That is, anti-epidermal growth factor monoclonal antibody was coated on a 96-well plate, and buffer (50 μL) and 200 μL of diluted serum were added. A standard to which human EGF (3.9-250 pg / mL) was added was used as a standard for quantification. After reacting at room temperature for 2 hours, it was washed 3 times with a buffer solution. Thereafter, an anti-horseradish peroxidase-labeled EGF polyclonal antibody (200 μL) was added and reacted at room temperature for 1 hour. Next, after washing 3 times with a buffer solution, a TMB solution (200 μL) is added, and the mixture is allowed to react for 20 minutes at room temperature, protected from light. The absorbance at wavelengths of 450 nm and 550 nm was measured with an automatic microplate reader (EL340I, BIO-TEC, USA). The EGF content in the specimen was measured by sandwich ELISA, and the EGF concentration was calculated from the calibration curve.
(3) Statistical analysis Data are shown as mean ± standard deviation. Statistical analysis between the two groups was analyzed with Mann-Whitney U-test. A p value of 0.05 or less was considered statistically significant.
(4) Results i) Concentration of serum EGF in all subjects FIG. 3 shows the state of spraying of serum EGF concentration in normal controls and subjects with high-functioning autism.

As seen in the above experimental results, the serum EGF concentration (average value: 80.96 pg / ml) of patients with high-functioning autism is the concentration of normal controls in the same age group (average value: 315.1 ng / ml) It was found to be significantly lower than Since EGF plays an extremely important role in the developmental process of the brain, the decreased serum EGF level is thought to contribute to the pathophysiology of high-functioning autism. For example, a patient's serum EGF value is the average value of serum EGF values of healthy subjects minus the value of standard deviation (191.5 pg /
If lower than (ml), it is diagnosed with high functioning autism.

  In short, this experiment confirmed that EGF plays an extremely important role in the pathophysiology of autism, and blood EGF is useful as a biological marker of autism.

  The present invention provides an early, simple and reliable diagnostic means of autism.

FIG. 3 is a scatter diagram of serum TGFβ1 concentration in normal control and autistic patients according to Example 1. It is a scatter diagram of the serum HGF density | concentration in the patient of a normal control and autism based on Example 2. FIG. FIG. 6 is a scatter diagram of serum EGF concentrations in normal control and autistic patients according to Example 3.

Claims (14)

A diagnostic agent for autism mainly composed of anti-transforming growth factor beta 1 antibody. The diagnostic agent for autism according to claim 1, wherein the diagnostic agent for autism is used for measuring a transforming growth factor beta 1 (TGFβ1) concentration in a human blood sample. The anti-transforming growth factor beta 1 antibody comprises an unlabeled anti-transforming growth factor beta 1 antibody and / or a labeled anti-transforming growth factor beta 1 antibody. A diagnostic agent for autism. An autism diagnostic kit comprising the diagnostic agent for autism according to any one of claims 1 to 3. Using the diagnostic agent for autism according to any one of claims 1 to 3 or the autism diagnostic kit according to claim 4, the transforming growth factor beta 1 concentration in a human blood sample is measured. Autism testing method. A test method for examining the effect of a therapeutic agent for autism using the diagnostic agent for autism according to claim 1 or the diagnostic kit for autism according to claim 4. The autism diagnostic agent according to any one of claims 1 to 3, wherein the autism diagnostic agent contains an anti-hepatocyte growth factor antibody and / or an anti-epidermal growth factor antibody. The diagnostic agent for autism is used for measuring transforming growth factor beta 1 concentration, hepatocyte growth factor (HGF) concentration and / or epidermal growth factor (EGF) concentration in a human blood sample The diagnostic agent for autism according to claim 7. The diagnostic agent for autism according to claim 7 or 8, wherein the anti-hepatocyte growth factor antibody comprises an unlabeled anti-hepatocyte growth factor antibody and / or a labeled anti-hepatocyte growth factor antibody. The diagnostic agent for autism according to claim 7 or 8, wherein the anti-epidermal growth factor antibody comprises an unlabeled anti-epidermal growth factor antibody and / or a labeled anti-epidermal growth factor antibody. The autism diagnostic kit comprised including the autism diagnostic agent in any one of Claims 7-10. A diagnostic agent for autism according to any one of claims 1 to 3, and an agent mainly comprising an anti-hepatocyte growth factor antibody and / or an agent mainly comprising an anti-epidermal growth factor antibody. Autism diagnostic kit. Using the autism diagnostic agent according to any one of claims 7 to 10, or the autism diagnosis kit according to claim 11 or claim 12, the transforming growth factor beta 1 concentration in a human blood sample and Autism assay method for measuring hepatocyte growth factor concentration and / or epidermal growth factor concentration. An assay method for testing the effect of a therapeutic agent for autism using the diagnostic agent for autism according to any one of claims 7 to 10, or the autism diagnostic kit according to claim 11 or claim 12. .

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