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WO2021154002A1 - Biomarker for diagnosis of parkinson's disease and method for diagnosis of parkinson's disease using same - Google Patents

Biomarker for diagnosis of parkinson's disease and method for diagnosis of parkinson's disease using same Download PDF

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Publication number
WO2021154002A1
WO2021154002A1 PCT/KR2021/001132 KR2021001132W WO2021154002A1 WO 2021154002 A1 WO2021154002 A1 WO 2021154002A1 KR 2021001132 W KR2021001132 W KR 2021001132W WO 2021154002 A1 WO2021154002 A1 WO 2021154002A1
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Prior art keywords
disease
parkinson
atg4b
protein
autoantibody
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PCT/KR2021/001132
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French (fr)
Korean (ko)
Inventor
박치후
박일홍
소병택
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Yep Bio Co Ltd
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Yep Bio Co Ltd
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Priority to CN202180026662.4A priority Critical patent/CN115398239A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to a biomarker for diagnosing Parkinson's disease, and a method for diagnosing Parkinson's disease using the same.
  • Parkinson's disease is a chronic disease caused by a lack of a neurotransmitter called dopamine in the brain, with main symptoms such as tremor (tremor), rigidity, tremor (action slow), and maintenance of unstable posture. It is a disease with pathophysiological symptoms such as a decrease in brain volume, aggregation of ⁇ -synuclein ( ⁇ Syn), etc., starting with deformation of the substantia nigra pars compacta of the midbrain Shows tremors, rigid behavior, etc.
  • Examples of tests performed in connection with the diagnosis of Parkinson's disease include PET scans, MRI scans, and tests for medical diseases.
  • Brain dopamine transporter PET (positron emission tomography) test is a test that can determine whether dopaminergic cells are damaged. symptoms can be identified. Drug-induced Parkinson's syndrome, vascular Parkinson's syndrome, Parkinson's symptoms accompanying Alzheimer's disease, and Parkinson's symptoms accompanying essential tremor sometimes show tremors similar to Parkinson's symptoms, but dopaminergic neurons are normal. there is.
  • Parkinson's symptoms it is important to distinguish it from diseases similar to Parkinson's disease, and to distinguish it from secondary Parkinson's syndrome and atypical Parkinson's syndrome, brain magnetic resonance imaging (MRI) is required first.
  • MRI brain magnetic resonance imaging
  • MRI is normal, while other diseases show characteristic MRI findings.
  • the methods for diagnosing Parkinson's disease are expensive and have complicated diagnostic procedures.
  • the number of patients suffering from Parkinson's disease increases year by year, and the therapeutic agent market is growing in proportion to this, but the development of the Parkinson's disease diagnosis market is still insufficient.
  • autoantibodies are universally present in all human blood, and it is known that the amount of autoantibodies increases with age.
  • Autoantibodies are known to be involved in the adaptive debris clearance mechanism as self-reactive antibodies. For example, in the case of an autoimmune disease, it is known that the disease is caused or aggravated by an autoantibody against one's own specific cells or tissue components.
  • autoantibodies and Parkinson's disease are closely related. Accordingly, autoantibodies are attracting attention as biomarkers for the diagnosis and prediction of Alzheimer's disease.
  • the present inventors attempted to discover autoantibodies capable of predicting the diagnosis and onset of Parkinson's disease from the patient's blood, profiling the autoantibody from the blood of the Parkinson's disease patient, and selecting and verifying the Parkinson's disease-specific autoantibody.
  • the present invention relates to a composition for diagnosing Parkinson's disease, a biomarker for diagnosing Parkinson's disease, and Parkinson's disease using the ATG4B protein that specifically binds to the autoantibody in order to measure the level of autoantibody against the cysteine protease ATG4B (ATG4B) protein to provide a diagnostic method.
  • ATG4B protein that specifically binds to the autoantibody in order to measure the level of autoantibody against the cysteine protease ATG4B (ATG4B) protein to provide a diagnostic method.
  • composition for diagnosing Parkinson's disease includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • the ATG4B protein may include the amino acid sequence shown in SEQ ID NO: 1.
  • kits for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.
  • kit can detect an autoantibody to the cysteine protease ATG4B (ATG4B) protein.
  • the detection of the autoantibody is ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmnoassay), sandwich assay (Sandwich assay), Western blot (Western Blot), immunoblot analysis (Immunoblot assay), and immunohistochemical staining method (Immnohistochemical staining) may be performed with one or more selected from the group consisting of.
  • an information providing method for diagnosing Parkinson's disease comprising: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.
  • ATG4B autoantibody to cysteine protease ATG4B
  • the biological sample may be selected from the group consisting of blood, plasma, and serum.
  • the autoantibody level against the ATG4B protein can be measured using an antigen-antibody reaction with the autoantibody.
  • an antigen-antibody reaction is performed by adding a biological sample isolated from a subject to a fixed body coated with cysteine protease ATG4B (ATG4B) protein. making; detecting the antigen-antibody reaction produced through the above step using a chromogenic substrate solution; And it may include the step of determining that the subject has Parkinson's disease or is highly likely to have the detection amount compared to the normal subject.
  • ATG4B cysteine protease ATG4B
  • the fixture may be selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass.
  • the method comprising: measuring the level of an autoantibody to ATG4B (cysteine protease ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject.
  • ATG4B cyste protease ATG4B
  • the present invention provides a biomarker for diagnosing Parkinson's disease by confirming that the autoantibody level of ATG4B (cysteine protease ATG4B) protein is high in the Parkinson's disease patient group. By screening in advance, it can be very usefully used to provide a basis for early diagnosis and treatment of Parkinson's disease.
  • ATG4B cyste protease ATG4B
  • the information providing method for diagnosing Parkinson's disease according to the present invention can diagnose Parkinson's disease more accurately and early by detecting the autoantibody of the ATG4B protein.
  • Example 1 shows an autoantibody reaction method for Parkinson's disease patients and normal groups using 6000 protein chips (Example 1).
  • Figure 2 is a graph showing the protein increased in the Parkinson's disease patient group (Example 1).
  • Figure 3 is a graph showing the protein decreased in the Parkinson's disease patient group (Example 1).
  • Example 4 is a graph showing the protein showing different aspects in the Advanced, Early patient groups (Example 1).
  • Example 5 is a schematic diagram of the Mini-Chip manufactured in Example 2.
  • Example 6 is a table showing the names of proteins applied in Example 2.
  • FIG. 7 is a graph showing a pilot screening result of Example 2.
  • Example 8 is a graph showing the results of the secondary screening of Example 3.
  • composition for diagnosing Parkinson's disease includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • kits for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.
  • an information providing method for diagnosing Parkinson's disease comprising: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.
  • ATG4B autoantibody to cysteine protease ATG4B
  • the present inventors tried to discover autoantibodies capable of diagnosing and predicting the onset of Parkinson's disease from the patient's blood, profiling the autoantibody from the blood of the Parkinson's disease patient, and selecting and verifying the Parkinson's disease-specific autoantibody.
  • autoantibodies is a type of antibody and refers to an antibody produced by an immune response to an individual's own protein. It is known that many autoimmune diseases produce such autoantibodies. A single type of autoantibody test is not diagnostic in itself, but may provide clues as to whether a particular disease is likely or not present. Each autoantibody outcome should be considered individually or as part of the overall medical history.
  • diagnosis refers to determining an individual's susceptibility to a particular disease or disorder, determining whether an individual currently has a particular disease or disorder, or providing information on the efficacy of treatment. This includes monitoring the condition of the object for harm. Diagnosis for the purpose of the present invention is to determine whether or not Parkinson's disease is onset or the stage of onset of Parkinson's disease.
  • composition for diagnosing Parkinson's disease includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.
  • composition for diagnosing Parkinson's disease of the present invention uses the significant difference in the expression of the autoantibody to the ATG4B protein in Parkinson's disease patients compared to normal individuals to obtain useful information for diagnosing or predicting the prognosis of Parkinson's disease. will be. This has not been known so far, and was first discovered by the present invention, and has great significance in that it is possible to diagnose or predict Parkinson's disease with high sensitivity and specificity.
  • the ATG4B protein refers to the cysteine protease ATG4B isoform of NCBI Reference Sequence: NP_037457.3.
  • the ATG4B protein may include the amino acid sequence represented by SEQ ID NO: 1.
  • kits for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.
  • the kit for diagnosing Parkinson's disease of the present invention can detect an autoantibody to the cysteine protease ATG4B (ATG4B) protein.
  • the kit for diagnosing Parkinson's disease of the present invention may be carried out in an antigen-antibody reaction method in which an ATG4B protein autoantibody is used as a target antigen and specifically binds to the ATG4B protein autoantibody.
  • the antigen-antibody reaction can be carried out using an antibody or aptamer that specifically binds to an autoantibody of the ATG4B protein of the present invention.
  • the antigen used in the present invention may be prepared as an ATG4B protein by methods commonly practiced in the art, for example, a fusion method or a recombinant DNA method.
  • Detection of the autoantibody in the diagnostic kit of the present invention is performed by ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmnoassay), Sandwich assay, Western Blot, Immunoblot assay, and immunity It may be performed by at least one selected from the group consisting of histochemical staining (Immnohistochemical staining).
  • the kit includes a fixture for immobilizing the ATG4B protein or a fragment thereof; a secondary antibody conjugate in which a chromophore that specifically binds to the autoantibody against ATG4B is conjugated; It may include a chromogenic substrate solution to be subjected to a color reaction with the label, a washing solution, and an enzyme reaction stop solution.
  • a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, and a glass slide glass, etc. this can be used as a fixture for the antigen-antibody binding reaction.
  • the label of the secondary antibody is preferably a conventional color developer for a color reaction, and HRP (Horseradish peroxidase), alkaline phosphatase, colloid gold (Coloid gold), FITC (Poly L-lysine-fluorescein isothiocyanate) ), a fluorescent substance (Fluorescein) such as RITC (Rhodamine-B-isothiocyanate), and a label such as a dye (Dye) may be used.
  • HRP Haseradish peroxidase
  • alkaline phosphatase colloid gold
  • FITC Poly L-lysine-fluorescein isothiocyanate
  • Fluorescein fluorescent substance
  • RITC Rhodamine-B-isothiocyanate
  • a label such as a dye (Dye)
  • the substrate inducing the color development according to the marker for the color reaction
  • TMB 3,3',5,5'-tetramethyl bezidine
  • ABTS 2,2'-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid)]
  • OPD ophenylenediamine
  • Chemiluminescence is generally used, and at this time, a chromogenic substrate such as Luminol is decomposed by HRP used as a label of the secondary antibody conjugate to generate 3-aminophthalate, and the degree of the luminescence is measured with light with a wavelength of 425 nm. By confirming, the presence or absence of autoantibody of ATG4B protein is detected.
  • the washing solution preferably contains phosphate buffer, NaCl and Tween 20, more preferably a buffer solution (PBST) composed of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (Tween 20). do.
  • PBST buffer solution
  • Tween 20 0.05% Tween 20
  • a method for providing information for diagnosing Parkinson's disease comprises the steps of: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.
  • ATG4B autoantibody to cysteine protease ATG4B
  • the term "individual” or “subject” refers to a subject for diagnosing Parkinson's disease or predicting the prognosis of Parkinson's disease.
  • the subject may be included without limitation as long as it is an animal that can develop Parkinson's disease, such as humans, dogs, horses, cattle, mice, goats, rabbits, chickens, ducks, and geese.
  • biological sample includes, but is not limited to, tissues, cells, whole blood, serum, plasma, and other samples such as saliva, sputum, cerebrospinal fluid or urine isolated from a subject suspected of having Parkinson's disease. can do.
  • the biological sample may be selected from the group consisting of blood, plasma, and serum.
  • normal individual refers to an individual not diagnosed with Parkinson's disease, and the level of autoantibody of the protein in a sample isolated from a normal individual and a sample isolated from an individual for which the diagnosis or prognosis of Parkinson's disease is to be predicted. By measuring and comparing , it is possible to accurately predict whether an individual suspected of having Parkinson's disease develops Parkinson's disease or the prognosis of Parkinson's disease.
  • the autoantibody level against the ATG4B protein can be measured using an antigen-antibody reaction with the autoantibody.
  • the antigen-antibody reaction may include an enzyme immunoassay (ELISA), immunoprecipitation method, fluorescence immunoassay, enzyme substrate coloring method, antigen-antibody aggregation method, and the like.
  • the level of autoantibody to the ATG4B protein measured in a biological sample isolated from a subject suspected of having Parkinson's disease in a sample isolated from a normal subject If it is significantly higher or lower than the measured level, it may be determined that the risk of developing Parkinson's disease is high or the prognosis is poor.
  • an antigen-antibody reaction is performed by adding a biological sample isolated from a subject to a fixed body coated with cysteine protease ATG4B (ATG4B) protein. making; detecting the antigen-antibody reaction produced through the above step using a chromogenic substrate solution; And it may include the step of determining that the subject has Parkinson's disease or is highly likely to have the detection amount compared to the normal subject.
  • ATG4B cysteine protease ATG4B
  • the substrate inducing the color development according to the marker for the color reaction
  • TMB 3,3',5,5'-tetramethyl bezidine
  • ABTS 2,2'-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid)]
  • OPD ophenylenediamine
  • Chemiluminescence is generally used, and at this time, a chromogenic substrate such as Luminol is decomposed by HRP used as a label of the secondary antibody conjugate to generate 3-aminophthalate, and the degree of the luminescence is measured with light with a wavelength of 425 nm. By confirming, the presence or absence of autoantibody of ATG4B protein is detected.
  • the fixture may be selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass.
  • recombinant proteins with GST tags were planted on a glass plate to prepare a protein chip. After that, serum from two of each of the normal group, early stage patient, and advanced stage patient was added to PBST buffer (PBS, 0.5% tween 20, pH 7.4) at a ratio of 1:2000 and reacted with the protein chip at room temperature for 2 hours. to bind to the antigen.
  • PBST buffer PBS, 0.5% tween 20, pH 7.4
  • the hIgG-488 (green fluorescence) secondary antibody (Invitrogen, A-10530) was reacted at a ratio of 1:5000 to confirm the presence of autoantibody in the patient's serum.
  • the GST tag of the implanted protein was used.
  • the GST antibody (Santa cruz, sc-33613) was reacted at room temperature for 2 hours, and after the same wash process, the amount of protein implanted in the protein chip was measured using a secondary antibody (Invitrogen, A-32740) of rlgG-594 (red fluorescence). .
  • a microarray reader was used to measure the antigen-antibody reaction of the protein chip through green and red fluorescence.
  • the patient information of the primary screening is shown in Table 1 below.
  • the recombinant protein purification vector (pDEST15) was transformed into the BL21 E. coli strain. 100ml of the BL21 E. coli strain containing the vector was grown on a shaker at 37°C so that the optical density (O.D.) reached 0.6, and then IPTG was added so that the final concentration was 0.4 mM. After 3 hours of induction at 37°C, BL21 was harvested, and cell lytic B buffer (0.5% cell lytic B, 0.1% TritonX-100, 0.07% ⁇ -ME, 1X Dnase1, protease inhibitor, lysozyme in PBST) was used at room temperature for 10 It was lysed by reacting for minutes. Thereafter, the GST-labeled recombinant protein was reacted with 100 ⁇ l of GST antibody-attached beads (GE, 17075601) at 4° C. for 3 hours, followed by pull-down of the desired protein.
  • GST-labeled recombinant protein was
  • wash buffer 1 50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol.
  • 0.1% Triton X-100, 1 mM PMSF, 0.07% tor washed 3 times, and then washed 3 times with Wash buffer 2 (50 mM Hepes pH 7.5, 100 mM NaCl, 1 mM EGTA, 10% glycerol) .
  • a protein purification vector was prepared using the Gateway cloning technique for 27 previously secured clones.
  • the 27 entry clones were transferred to the pDEST15 vector using LR clonase (Invitrogen, 11791020), and the protein was purified using the Cell lytic B method.
  • the patient's serum was reacted at room temperature at a ratio of 1:2000 for 2 hours so that the autoantibody in the serum was bound to the antigen protein.
  • the autoantigen antibody response was quantified through the chemiluminescence intensity shown through the HRP reaction.
  • HRP reaction For normalization of the entire experiment, a positive control group and antigen protein amount were used. i) As a positive control, hlgG was applied in the same amount to quantify the amount, ii) the amount of the applied protein was confirmed by GST tag and antibody reaction, This value was used to quantify the value.
  • the patient information of the pilot screening is shown in Table 2 below.
  • the same amount of GST protein and GST-ATG4B protein was applied to the nitrocellulose membrane, and the serum of the normal group and the patient group was reacted at room temperature at a ratio of 1:2000 for 2 hours, followed by a Chemiluminescence reaction using hIgG-HRP. The results were confirmed, and the application of the same amount of GST and GST-ATG4B was confirmed using the GST antibody.
  • the patient information of the secondary screening is shown in Table 3 below.
  • Example 1 Results of primary screening using a microarray using a protein chip
  • 1 shows the autoantibody reaction method of Parkinson's disease patients and normal groups using 6000 protein chips.
  • the 14 proteins found to be decreased in the patient group were STK24, NDUFV2, FLJ37087, ANXA2, LACTTB2, CLIC1, MSRA, YWHAE, ACOX1, G6PD, MRPS36, ATP5L, HSPA8, and ZNF516. was confirmed (Fig. 3).
  • Proteins that decreased in the early stage but increased in the advanced stage were SF3B4, SOD1, MPP1, DHODH, PECI, ZYX, ATG4B, OGG1, CBX1, GRHPR, and FHL3.
  • SMARCA1 the amount increased in the early stage, but showed a decreasing pattern in the advanced PD patients (Fig. 4).
  • ATG4B protein as a protein involved in cellular autophagy, its function is known to be changed in many degenerative brain diseases including Parkinson's disease.
  • the validation procedure was performed on more than 30 patient groups.

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Abstract

The present invention provides a composition for diagnosis of Parkinson's disease, comprising a cysteine protease ATG4B (ATG4B) protein that specifically binds to an autoantibody in order to measure the level of the autoantibody against the ATG4B protein; a biomarker for diagnosing Parkinson's disease; and a method for diagnosing Parkinson's disease using same. The present invention has found that a high level of the autoantibody against the ATG4B protein is observed in a Parkinson's disease patient group, and based thereon provides a biomarker for the detection of Parkinson's disease, which can be very useful in early screening of individuals at high risk of developing Parkinson's disease in the future and patients with Parkinson's disease, thus providing the basis that enables early diagnosis and treatment of Parkinson's disease. In addition, a method for providing information for the diagnosis of Parkinson's disease according to the present invention enables more accurate detection of Parkinson's disease in early stages by detecting an autoantibody to the ATG4B protein.

Description

파킨슨병 진단용 바이오마커 및 이를 이용한 파킨슨병 진단 방법Biomarker for diagnosing Parkinson's disease and method for diagnosing Parkinson's disease using the same

본 발명은 파킨슨병 진단용 바이오마커, 및 이를 이용한 파킨슨병 진단 방법에 관한 것이다.The present invention relates to a biomarker for diagnosing Parkinson's disease, and a method for diagnosing Parkinson's disease using the same.

파킨슨병은 진전(떨림), 경직, 서동증(행동이 느려짐), 불안정한 자세 유지 등을 주증상으로 하는 병으로 뇌에서 도파민이라는 신경전달물질이 부족하게 되어 생기는 만성질환이고, 중추신경계 퇴행성질환의 하나이며, 중뇌의 흑색질 밀집부(Substantia nigra pars compacta)의 변형을 시작으로 뇌의 부피의 감소, 알파-시누클레인(α-synuclein, αSyn)의 응집 등을 병태생리적 증상으로 갖는 질환으로 보행의 불완전, 손떨림, 강직된 행동 등을 보인다.Parkinson's disease is a chronic disease caused by a lack of a neurotransmitter called dopamine in the brain, with main symptoms such as tremor (tremor), rigidity, tremor (action slow), and maintenance of unstable posture. It is a disease with pathophysiological symptoms such as a decrease in brain volume, aggregation of α-synuclein (αSyn), etc., starting with deformation of the substantia nigra pars compacta of the midbrain Shows tremors, rigid behavior, etc.

이러한 파킨슨병의 진단과 관련하여 수행되는 검사로는 PET 검사, MRI 검사, 내과적 질환에 대한 검사 등이 있다.Examples of tests performed in connection with the diagnosis of Parkinson's disease include PET scans, MRI scans, and tests for medical diseases.

뇌 도파민 운반체 양전자 단층 촬영(dopamine transporter PET, positron emission tomography) 검사는 도파민성 세포의 손상 여부를 알 수 있는 검사로서, 뇌 도파민 운반체 양전자 단층 촬영을 하게 되면 파킨슨병 때문이 아닌 다른 원인들에 의한 파킨슨증상을 확인할 수 있다. 약물 유발성 파킨슨증후군, 혈관성 파킨슨증후군, 알츠하이머병에서 동반되는 파킨슨증상, 본태성 진전에서 동반되는 파킨슨증상의 경우 일견 파킨슨증상과 유사한 떨림 등을 보이는 경우가 있으나, 도파민성 신경세포는 정상임을 확인할 수 있다.Brain dopamine transporter PET (positron emission tomography) test is a test that can determine whether dopaminergic cells are damaged. symptoms can be identified. Drug-induced Parkinson's syndrome, vascular Parkinson's syndrome, Parkinson's symptoms accompanying Alzheimer's disease, and Parkinson's symptoms accompanying essential tremor sometimes show tremors similar to Parkinson's symptoms, but dopaminergic neurons are normal. there is.

파킨슨 증상을 보이는 경우에 파킨슨병과 유사한 질환들과 구분하는 것이 중요하고, 이차성 파킨슨증후군 및 비전형성 파킨슨증후군 등과 구분하기 위해서는 먼저 뇌 자기공명영상(MRI) 검사가 필요하다. 파킨슨병의 경우에는 MRI가 정상소견인 반면에 다른 질환들은 특징적인 MRI 소견을 보인다.In the case of Parkinson's symptoms, it is important to distinguish it from diseases similar to Parkinson's disease, and to distinguish it from secondary Parkinson's syndrome and atypical Parkinson's syndrome, brain magnetic resonance imaging (MRI) is required first. In the case of Parkinson's disease, MRI is normal, while other diseases show characteristic MRI findings.

파킨슨병 진단 방법 중 내과적 질환에 대한 검사(혈액검사, 소변 검사, 심전도, 가슴 엑스선 검사)방법은 내과적 질환이 전신 위약감을 일으키면서, 파킨슨 증상으로 오인하는 경우가 있는데 이를 확인하기 위해서는 다른 내과적인 질환이 없는지 확인하기 위한 검사가 필요하다.Among the methods of diagnosing Parkinson's disease, the examination (blood test, urinalysis, electrocardiogram, chest X-ray) for medical diseases is often mistaken for Parkinson's symptoms while medical diseases cause systemic weakness. Tests are needed to confirm that there are no chronic diseases.

상기 파킨슨병 진단 방법들은 고가이고 진단 절차가 복잡한 문제가 있다. 또한, 파킨슨병을 앓고 있는 환자는 해마다 크게 증가하고 치료제 시장도 이에 비례하여 크게 성장하고 있지만, 정작 파킨슨병 진단 시장의 발전은 미진한 실정이다.The methods for diagnosing Parkinson's disease are expensive and have complicated diagnostic procedures. In addition, the number of patients suffering from Parkinson's disease increases year by year, and the therapeutic agent market is growing in proportion to this, but the development of the Parkinson's disease diagnosis market is still insufficient.

따라서, 파킨슨병을 그 유사질환과 구분하여 신속하고 경제적으로 진단할 수 있는 기술 및 이와 동시에 치료를 병행할 수 있는 기술에 대한 개발이 시급한 상황이다.Therefore, there is an urgent need to develop a technology capable of rapidly and economically diagnosing Parkinson's disease by distinguishing it from its similar diseases, and a technology capable of simultaneously treating Parkinson's disease.

한편, 자가항체(autoantibody)는 모든 인간의 혈액에서 보편적으로 존재하며, 나이가 들어감에 따라 자가항체의 양이 증가하는 것으로 알려져 있다. 자가항체는 자기 자신에 대항하는(self-reactive) 항체로서 adaptive debrisclearance mechanism에 관여한다고 알려져 있다. 예를 들어, 자가면역질환의 경우 자기 자신의 특정 세포나 조직 구성물에 대항하는 자가항체에 의해서 질환이 발생하거나 악화된다고 알려져 있다.On the other hand, autoantibodies are universally present in all human blood, and it is known that the amount of autoantibodies increases with age. Autoantibodies are known to be involved in the adaptive debris clearance mechanism as self-reactive antibodies. For example, in the case of an autoimmune disease, it is known that the disease is caused or aggravated by an autoantibody against one's own specific cells or tissue components.

최근 자가항체와 파킨슨병이 밀접하게 연관되어 있음이 알려졌다. 이에 자가항체가 알츠하이머 질환의 진단과 발병예측을 위한 바이오마커로 주목을 받고 있다.Recently, it has been known that autoantibodies and Parkinson's disease are closely related. Accordingly, autoantibodies are attracting attention as biomarkers for the diagnosis and prediction of Alzheimer's disease.

이에, 본 발명자들은 환자의 혈액으로부터 파킨슨병의 진단 및 발병 예측이 가능한 자가항체를 발굴하고자 하였으며, 파킨슨병 환자의 혈액으로부터 자가항체를 프로파일링하고 파킨슨병 특이적인 자가항체를 선별하여 검증하였다.Accordingly, the present inventors attempted to discover autoantibodies capable of predicting the diagnosis and onset of Parkinson's disease from the patient's blood, profiling the autoantibody from the blood of the Parkinson's disease patient, and selecting and verifying the Parkinson's disease-specific autoantibody.

본 발명은 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하기 위하여 상기 자가항체에 특이적으로 결합하는 ATG4B 단백질을 포함하는 파킨슨병 진단용 조성물, 파킨슨병 진단용 바이오마커, 및 이를 이용한 파킨슨병 진단 방법을 제공하는 것이다.The present invention relates to a composition for diagnosing Parkinson's disease, a biomarker for diagnosing Parkinson's disease, and Parkinson's disease using the ATG4B protein that specifically binds to the autoantibody in order to measure the level of autoantibody against the cysteine protease ATG4B (ATG4B) protein to provide a diagnostic method.

본 발명의 일 측면에 따른 파킨슨병 진단용 조성물은, ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하기 위하여, 상기 자가항체에 특이적으로 결합하는 ATG4B 단백질을 포함한다.The composition for diagnosing Parkinson's disease according to an aspect of the present invention includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.

그리고, 상기 ATG4B 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함할 수 있다.And, the ATG4B protein may include the amino acid sequence shown in SEQ ID NO: 1.

본 발명의 일 측면에 따르면 상기 파킨슨병 진단용 조성물을 포함하는 파킨슨병 진단용 키트가 제공된다.According to one aspect of the present invention, there is provided a kit for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.

그리고, 상기 키트는 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체(autoantibody)를 검출할 수 있다.In addition, the kit can detect an autoantibody to the cysteine protease ATG4B (ATG4B) protein.

또한, 상기 자가항체의 검출은 ELISA(Enzyme-linked immunosorbent assay), RIA(Radioimmnoassay), 샌드위치 측정법(Sandwich assay), 웨스턴 블롯(Western Blot), 면역블롯 분석(Immunoblot assay), 및 면역조직화학염색 방법(Immnohistochemical staining)으로 이루어지는 군으로부터 선택되는 하나 이상으로 수행될 수 있다.In addition, the detection of the autoantibody is ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmnoassay), sandwich assay (Sandwich assay), Western blot (Western Blot), immunoblot analysis (Immunoblot assay), and immunohistochemical staining method (Immnohistochemical staining) may be performed with one or more selected from the group consisting of.

본 발명의 다른 측면에 따른 파킨슨병 진단을 위한 정보제공 방법은, 피검체로부터 분리된 생물학적 시료로부터 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하는 단계; 상기 측정된 자가항체 수준을 정상인 개체로부터 분리된 생물학적 시료로부터 측정된 ATG4B 단백질에 대한 자가항체 수준과 비교하는 단계: 및 상기 피검체의 자가항체 수준이 상기 정상인 개체의 자가항체 수준보다 높은 경우 파킨슨병 양성으로 판정하는 단계를 포함할 수 있다.According to another aspect of the present invention, there is provided an information providing method for diagnosing Parkinson's disease, comprising: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.

그리고, 상기 생물학적 시료는 혈액, 혈장, 및 혈청으로 이루어진 군으로부터 선택될 수 있다.In addition, the biological sample may be selected from the group consisting of blood, plasma, and serum.

또한, 상기 ATG4B 단백질에 대한 자가항체 수준은 상기 자가항체와의 항원-항체 반응을 이용하여 측정될 수 있다.In addition, the autoantibody level against the ATG4B protein can be measured using an antigen-antibody reaction with the autoantibody.

본 발명의 또 다른 측면에 따른 파킨슨병 진단 정보를 제공하기 위해 자가 항체를 검출하는 방법은, ATG4B(cysteine protease ATG4B) 단백질이 코팅된 고정체에 피검체로부터 분리된 생물학적 시료를 가하여 항원-항체 반응시키는 단계; 상기 단계를 통해 생성된 항원-항체 반응물을 발색기질 용액을 이용하여 검출하는 단계; 및 상기 검출량이 정상인 개체에 비해 증가된 피검체를 파킨슨병에 걸렸거나 걸릴 가능성이 높은 것으로 판정하는 단계를 포함할 수 있다.In a method for detecting autoantibodies to provide Parkinson's disease diagnostic information according to another aspect of the present invention, an antigen-antibody reaction is performed by adding a biological sample isolated from a subject to a fixed body coated with cysteine protease ATG4B (ATG4B) protein. making; detecting the antigen-antibody reaction produced through the above step using a chromogenic substrate solution; And it may include the step of determining that the subject has Parkinson's disease or is highly likely to have the detection amount compared to the normal subject.

그리고, 상기 고정체는 니트로셀룰로오스 막, PVDF 막, 폴리비닐 또는 폴리스티렌 수지로 합성된 웰 플레이트, 및 유리로 된 슬라이드 글라스로 이루어지는 군으로부터 선택될 수 있다.In addition, the fixture may be selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass.

본 발명의 다른 측면에 따르면, 피검체로부터 분리된 생물학적 시료로부터 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하는 단계; 상기 측정된 자가항체 수준을 정상인 개체로부터 분리된 생물학적 시료로부터 측정된 ATG4B 단백질에 대한 자가항체 수준과 비교하는 단계: 및 상기 피검체의 자가항체 수준이 상기 정상인 개체의 자가항체 수준보다 높은 경우 파킨슨병 양성으로 판정하는 단계를 포함하는 파킨슨병 진단방법이 제공된다.According to another aspect of the present invention, the method comprising: measuring the level of an autoantibody to ATG4B (cysteine protease ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. There is provided a method for diagnosing Parkinson's disease comprising the step of determining positive.

본 발명은, 파킨슨병 환자군에서 ATG4B(cysteine protease ATG4B) 단백질의 자가항체 수준이 높게 나타남을 확인하여 이에 대한 파킨슨병 진단용 바이오마커를 제공함으로써, 향후 파킨슨병으로 발병할 가능성이 높은 고위험군 및 발병 환자를 미리 선별하여, 파킨슨병을 조기에 진단하고 치료할 기반을 제공하는데 매우 유용하게 사용될 수 있다.The present invention provides a biomarker for diagnosing Parkinson's disease by confirming that the autoantibody level of ATG4B (cysteine protease ATG4B) protein is high in the Parkinson's disease patient group. By screening in advance, it can be very usefully used to provide a basis for early diagnosis and treatment of Parkinson's disease.

또한, 본 발명에 따른 파킨슨병 진단을 위한 정보제공 방법은 ATG4B 단백질의 자가항체를 검출함으로써 파킨슨병을 보다 정확하고 조기에 진단할 수 있다.In addition, the information providing method for diagnosing Parkinson's disease according to the present invention can diagnose Parkinson's disease more accurately and early by detecting the autoantibody of the ATG4B protein.

도 1은 6000개 단백질 칩을 이용한 파킨슨병 환자 및 정상군 Autoantibody 반응 방법을 나타낸 것이다 (실시예 1).1 shows an autoantibody reaction method for Parkinson's disease patients and normal groups using 6000 protein chips (Example 1).

도 2는 파킨슨병 환자군에서 증가하는 단백질을 나타낸 그래프이다 (실시예 1).Figure 2 is a graph showing the protein increased in the Parkinson's disease patient group (Example 1).

도 3은 파킨슨병 환자군에서 감소하는 단백질을 나타낸 그래프이다 (실시예 1).Figure 3 is a graph showing the protein decreased in the Parkinson's disease patient group (Example 1).

도 4는 Advanced, Early 환자군에서 서로 다른 양상이 나타난 단백질을 나타낸 그래프이다 (실시예 1).4 is a graph showing the protein showing different aspects in the Advanced, Early patient groups (Example 1).

도 5는 실시예 2에서 제작된 Mini-Chip의 모식도이다.5 is a schematic diagram of the Mini-Chip manufactured in Example 2.

도 6은 실시예 2에서 도포된 단백질명을 나타낸 표이다. 6 is a table showing the names of proteins applied in Example 2.

도 7은 실시예 2의 파일럿 스크리닝 결과를 나타낸 그래프이다.7 is a graph showing a pilot screening result of Example 2. FIG.

도 8은 실시예 3의 2차 스크리닝 결과를 나타낸 그래프이다.8 is a graph showing the results of the secondary screening of Example 3.

본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

본 발명의 일 측면에 따른 파킨슨병 진단용 조성물은, ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하기 위하여, 상기 자가항체에 특이적으로 결합하는 ATG4B 단백질을 포함한다.The composition for diagnosing Parkinson's disease according to an aspect of the present invention includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.

본 발명의 일 측면에 따르면 상기 파킨슨병 진단용 조성물을 포함하는 파킨슨병 진단용 키트가 제공된다.According to one aspect of the present invention, there is provided a kit for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.

본 발명의 다른 측면에 따른 파킨슨병 진단을 위한 정보제공 방법은, 피검체로부터 분리된 생물학적 시료로부터 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하는 단계; 상기 측정된 자가항체 수준을 정상인 개체로부터 분리된 생물학적 시료로부터 측정된 ATG4B 단백질에 대한 자가항체 수준과 비교하는 단계: 및 상기 피검체의 자가항체 수준이 상기 정상인 개체의 자가항체 수준보다 높은 경우 파킨슨병 양성으로 판정하는 단계를 포함할 수 있다.According to another aspect of the present invention, there is provided an information providing method for diagnosing Parkinson's disease, comprising: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.

이하 발명의 구체적인 구현예에 따른 파킨슨병 진단용 바이오마커, 및 이를 이용한 파킨슨병 진단 방법에 관하여 보다 상세하게 설명하기로 한다.Hereinafter, a biomarker for diagnosing Parkinson's disease and a method for diagnosing Parkinson's disease using the same according to specific embodiments of the present invention will be described in more detail.

종래 파킨슨병 진단 방법들은 고가이고 진단 절차가 복잡한 문제점이 있었고, 환자는 해마다 크게 증가하고 치료제 시장도 이에 비례하여 크게 성장하고 있지만, 파킨슨병 진단 시장의 발전은 미진한 실정이었다.Conventional methods for diagnosing Parkinson's disease have problems in that they are expensive and have complicated diagnostic procedures, and the number of patients increases year by year, and the market for therapeutic agents is growing in proportion to this.

이에 본 발명자들은 환자의 혈액으로부터 파킨슨병의 진단 및 발병 예측이 가능한 자가항체를 발굴하고자 하였으며, 파킨슨병 환자의 혈액으로부터 자가항체를 프로파일링하고 파킨슨병 특이적인 자가항체를 선별하여 검증하였다.Accordingly, the present inventors tried to discover autoantibodies capable of diagnosing and predicting the onset of Parkinson's disease from the patient's blood, profiling the autoantibody from the blood of the Parkinson's disease patient, and selecting and verifying the Parkinson's disease-specific autoantibody.

본 발명의 용어 “자가항체(autoantibodies)”는 항체의 일종으로 개체가 가지고 있는 개체 자신의 단백질에 대한 면역 반응에 의해 생성된 항체를 말한다. 많은 자가면역질환의 경우 이러한 자가항체를 생성하는 것으로 알려져 있다. 단일한 종류의 자가항체 검사는 그 자체로 진단적이지는 않으나 특정 질환이 존재할 가능성이 있는지 또는 없는지에 대한 단서를 제공할 수 있다. 각 자가항체 결과는 개별적으로 또는 전체 병력의 일부로서 고려되어야 한다.As used herein, the term “autoantibodies” is a type of antibody and refers to an antibody produced by an immune response to an individual's own protein. It is known that many autoimmune diseases produce such autoantibodies. A single type of autoantibody test is not diagnostic in itself, but may provide clues as to whether a particular disease is likely or not present. Each autoantibody outcome should be considered individually or as part of the overall medical history.

본 발명의 용어, "진단"은 특정 질병 또는 질환에 대한 개체의 감수성(susceptibility)을 판정하는 것, 개체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것 또는 치료 효능에 대한 정보를 제공하기 위해 개체의 상태를 모니터링 하는 것을 포함한다. 본 발명의 목적상 진단은 파킨슨병의 발병 여부 또는 파킨슨병의 발병 단계를 확인하는 것이다.As used herein, the term "diagnosis" refers to determining an individual's susceptibility to a particular disease or disorder, determining whether an individual currently has a particular disease or disorder, or providing information on the efficacy of treatment. This includes monitoring the condition of the object for harm. Diagnosis for the purpose of the present invention is to determine whether or not Parkinson's disease is onset or the stage of onset of Parkinson's disease.

본 발명의 일 측면에 따른 파킨슨병 진단용 조성물은, ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하기 위하여, 상기 자가항체에 특이적으로 결합하는 ATG4B 단백질을 포함한다.The composition for diagnosing Parkinson's disease according to an aspect of the present invention includes an ATG4B protein that specifically binds to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein.

본 발명의 파킨슨병 진단용 조성물은 파킨슨병 환자에서 정상 개체와 비교하여 상기 ATG4B 단백질에 대한 자가항체의 유의적인 발현 차이가 나타남을 이용하여 파킨슨병의 진단 또는 예후 예측을 위한 유용한 정보를 획득할 수 있는 것이다. 이는 지금까지 알려진 바 없으며, 본 발명에 의해 최초로 밝혀진 것으로, 높은 민감도 및 특이도로 파킨슨병 진단 또는 예후 예측이 가능한 점에서 그 의의가 크다.The composition for diagnosing Parkinson's disease of the present invention uses the significant difference in the expression of the autoantibody to the ATG4B protein in Parkinson's disease patients compared to normal individuals to obtain useful information for diagnosing or predicting the prognosis of Parkinson's disease. will be. This has not been known so far, and was first discovered by the present invention, and has great significance in that it is possible to diagnose or predict Parkinson's disease with high sensitivity and specificity.

본 발명에서 ATG4B 단백질은 NCBI Reference Sequence: NP_037457.3 인 cysteine protease ATG4B isoform 을 의미한다. 상기 ATG4B 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함할 수 있다.In the present invention, the ATG4B protein refers to the cysteine protease ATG4B isoform of NCBI Reference Sequence: NP_037457.3. The ATG4B protein may include the amino acid sequence represented by SEQ ID NO: 1.

본 발명의 일 측면에 따르면 상기 파킨슨병 진단용 조성물을 포함하는 파킨슨병 진단용 키트가 제공된다.According to one aspect of the present invention, there is provided a kit for diagnosing Parkinson's disease comprising the composition for diagnosing Parkinson's disease.

본 발명의 파킨슨병 진단용 키트는 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체(autoantibody)를 검출할 수 있다.The kit for diagnosing Parkinson's disease of the present invention can detect an autoantibody to the cysteine protease ATG4B (ATG4B) protein.

본 발명의 파킨슨병 진단용 키트는 ATG4B 단백질의 자가항체를 타겟 항원으로 하여, 상기 ATG4B 단백질의 자가항체와 특이적으로 결합하는 항원-항체 반응 방식으로 실시될 수 있다. 이 경우, 상기 항원-항체 반응은 본 발명의 ATG4B 단백질의 자가항체에 특이적으로 결합하는 항체 또는 앱타머를 이용하여 실시될 수 있다.The kit for diagnosing Parkinson's disease of the present invention may be carried out in an antigen-antibody reaction method in which an ATG4B protein autoantibody is used as a target antigen and specifically binds to the ATG4B protein autoantibody. In this case, the antigen-antibody reaction can be carried out using an antibody or aptamer that specifically binds to an autoantibody of the ATG4B protein of the present invention.

본 발명에서 이용되는 항원은 ATG4B 단백질로서 당업계에서 통상적으로 실시되는 방법들, 예를 들어, 융합 방법, 재조합 DNA 방법에 의해 제조될 수 있다.The antigen used in the present invention may be prepared as an ATG4B protein by methods commonly practiced in the art, for example, a fusion method or a recombinant DNA method.

본 발명의 진단용 키트에서 상기 자가항체의 검출은 ELISA(Enzyme-linked immunosorbent assay), RIA(Radioimmnoassay), 샌드위치 측정법(Sandwich assay), 웨스턴 블롯(Western Blot), 면역블롯 분석(Immunoblot assay), 및 면역조직화학염색 방법(Immnohistochemical staining)으로 이루어지는 군으로부터 선택되는 하나 이상으로 수행될 수 있다.Detection of the autoantibody in the diagnostic kit of the present invention is performed by ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmnoassay), Sandwich assay, Western Blot, Immunoblot assay, and immunity It may be performed by at least one selected from the group consisting of histochemical staining (Immnohistochemical staining).

상기 키트는 상기 ATG4B 단백질 또는 이의 단편 등을 고정하기 위한 고정체; 상기 ATG4B에 대한 자가항체와 특이적으로 결합하는 발색표지체가 컨쥬게이션된 2차 항체 접합체(Conjugate); 상기 표지체와 발색반응할 발색기질 용액, 세척액, 및 효소반응 정지용액을 포함할 수 있다.The kit includes a fixture for immobilizing the ATG4B protein or a fragment thereof; a secondary antibody conjugate in which a chromophore that specifically binds to the autoantibody against ATG4B is conjugated; It may include a chromogenic substrate solution to be subjected to a color reaction with the label, a washing solution, and an enzyme reaction stop solution.

상기 항원-항체 결합 반응을 위한 고정체로는 니트로셀룰로오스 막, PVDF막, 폴리비닐(Polyvinyl) 수지 또는 폴리스티렌(Polystyrene) 수지로 합성된 웰 플레이트 (Well plate) 및 유리로 된 슬라이드 글라스(Slide glass) 등이 사용될 수 있다.As a fixture for the antigen-antibody binding reaction, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, and a glass slide glass, etc. this can be used

상기 2차 항체의 표지체는 발색반응을 하는 통상의 발색제가 바람직하며, HRP(Horseradish peroxidase), 알칼리성 인산분해효소(Alkaline phosphatase), 콜로이드 골드(Coloid gold), FITC(Poly L-lysine-fluorescein isothiocyanate), RITC(Rhodamine-B-isothiocyanate) 등의 형광물질 (Fluorescein) 및 색소 (Dye) 등의 표지체가 사용될 수 있다.The label of the secondary antibody is preferably a conventional color developer for a color reaction, and HRP (Horseradish peroxidase), alkaline phosphatase, colloid gold (Coloid gold), FITC (Poly L-lysine-fluorescein isothiocyanate) ), a fluorescent substance (Fluorescein) such as RITC (Rhodamine-B-isothiocyanate), and a label such as a dye (Dye) may be used.

상기 발색을 유도하는 기질(발색기질)은 발색반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD(ophenylenediamine) 등을 사용할 수 있다.It is preferable to use the substrate (coloring substrate) inducing the color development according to the marker for the color reaction, TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (ophenylenediamine), and the like can be used.

보편적으로 화학발광법 (Chemiluminescence)이 주로 사용되며, 이때 Luminol과 같은 발색기질은 2차 항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 3-aminophthalate를 생성하고 그 발광체의 정도를 425nm 파장의 빛으로 확인함으로써 ATG4B 단백질의 자가항체 존재 유무를 검출한다.Chemiluminescence is generally used, and at this time, a chromogenic substrate such as Luminol is decomposed by HRP used as a label of the secondary antibody conjugate to generate 3-aminophthalate, and the degree of the luminescence is measured with light with a wavelength of 425 nm. By confirming, the presence or absence of autoantibody of ATG4B protein is detected.

세척액은 인산염 완충용액, NaCl 및 트윈 20(Tween 20)을 포함하는 것이 바람직하며, 0.02 M 인산염 완충용액, 0.13 M NaCl, 및 0.05% 트윈 20(Tween 20)으로 구성된 완충용액(PBST)이 더욱 바람직하다. 세척액은 항원-항체 결합반응 후 항원항체 결합체에 2차 항체를 반응시킨 다음 적당량을 고정체에 가하여 3 내지 6회 세척할 수 있다.The washing solution preferably contains phosphate buffer, NaCl and Tween 20, more preferably a buffer solution (PBST) composed of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (Tween 20). do. After the antigen-antibody binding reaction, the secondary antibody is reacted with the antigen-antibody conjugate, and an appropriate amount of the washing solution is added to the fixed body and washed 3 to 6 times.

본 발명에 따른 파킨슨병 진단을 위한 정보제공 방법은, 피검체로부터 분리된 생물학적 시료로부터 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하는 단계; 상기 측정된 자가항체 수준을 정상인 개체로부터 분리된 생물학적 시료로부터 측정된 ATG4B 단백질에 대한 자가항체 수준과 비교하는 단계: 및 상기 피검체의 자가항체 수준이 상기 정상인 개체의 자가항체 수준보다 높은 경우 파킨슨병 양성으로 판정하는 단계를 포함할 수 있다.A method for providing information for diagnosing Parkinson's disease according to the present invention comprises the steps of: measuring the level of an autoantibody to cysteine protease ATG4B (ATG4B) protein from a biological sample isolated from a subject; comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and Parkinson's disease when the autoantibody level of the subject is higher than the autoantibody level of the normal subject. It may include a step of determining positive.

본 발명의 용어, "개체" 또는 "피검체"는 파킨슨병을 진단하거나 파킨슨병의 예후를 예측하고자 하는 대상을 의미한다. 이때, 상기 개체는 사람을 비롯하여, 개, 말, 소, 쥐, 염소, 토끼, 닭, 오리, 거위 등의 파킨슨병이 발병될 수 있는 동물이라면 제한 없이 포함될 수 있다.As used herein, the term "individual" or "subject" refers to a subject for diagnosing Parkinson's disease or predicting the prognosis of Parkinson's disease. In this case, the subject may be included without limitation as long as it is an animal that can develop Parkinson's disease, such as humans, dogs, horses, cattle, mice, goats, rabbits, chickens, ducks, and geese.

본 발명의 용어, "생물학적 시료"란 이에 제한되는 것은 아니나, 파킨슨병 발병이 의심되는 개체로부터 분리된 조직, 세포, 전혈, 혈청, 혈장 외에도, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료 등을 포함할 수 있다. 특히, 본 발명에서 바람직하게는 상기 생물학적 시료는 혈액, 혈장, 및 혈청으로 이루어진 군으로부터 선택될 수 있다.As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, and other samples such as saliva, sputum, cerebrospinal fluid or urine isolated from a subject suspected of having Parkinson's disease. can do. In particular, in the present invention, preferably, the biological sample may be selected from the group consisting of blood, plasma, and serum.

본 발명의 용어, "정상인 개체"란 파킨슨병으로 진단되지 않은 개체를 의미하며, 정상인 개체로부터 분리된 시료와 파킨슨병의 진단 또는 예후를 예측하고자 하는 개체로부터 분리된 시료에서 상기 단백질의 자가항체 수준을 측정하고, 비교함으로써, 파킨슨병 발병이 의심되는 개체의 파킨슨병 발병 여부 또는 파킨슨병의 예후를 정확하게 예측할 수 있다.As used herein, the term "normal individual" refers to an individual not diagnosed with Parkinson's disease, and the level of autoantibody of the protein in a sample isolated from a normal individual and a sample isolated from an individual for which the diagnosis or prognosis of Parkinson's disease is to be predicted. By measuring and comparing , it is possible to accurately predict whether an individual suspected of having Parkinson's disease develops Parkinson's disease or the prognosis of Parkinson's disease.

또한, 상기 ATG4B 단백질에 대한 자가항체 수준은 상기 자가항체와의 항원-항체 반응을 이용하여 측정될 수 있다. 상기 항원-항체 반응은 효소면역측정법(ELISA), 면역침강법, 형광면역법, 효소기질발색법, 항원-항체응집법 등을 들 수 있다.In addition, the autoantibody level against the ATG4B protein can be measured using an antigen-antibody reaction with the autoantibody. The antigen-antibody reaction may include an enzyme immunoassay (ELISA), immunoprecipitation method, fluorescence immunoassay, enzyme substrate coloring method, antigen-antibody aggregation method, and the like.

본 발명의 파킨슨병 진단을 위한 정보를 제공하는 방법은, 파킨슨병 발병이 의심되는 개체로부터 분리된 생물학적 시료에서 측정된 ATG4B 단백질에 대한 자가항체의 수준을 측정한 결과, 정상인 개체로부터 분리된 시료에서 측정되는 수준 보다 유의적으로 높거나 낮은 경우, 파킨슨병의 발병 위험이 높거나, 그 예후가 나쁘다고 판정될 수 있다.In the method of providing information for diagnosing Parkinson's disease of the present invention, as a result of measuring the level of autoantibody to the ATG4B protein measured in a biological sample isolated from a subject suspected of having Parkinson's disease, in a sample isolated from a normal subject If it is significantly higher or lower than the measured level, it may be determined that the risk of developing Parkinson's disease is high or the prognosis is poor.

본 발명의 또 다른 측면에 따른 파킨슨병 진단 정보를 제공하기 위해 자가 항체를 검출하는 방법은, ATG4B(cysteine protease ATG4B) 단백질이 코팅된 고정체에 피검체로부터 분리된 생물학적 시료를 가하여 항원-항체 반응시키는 단계; 상기 단계를 통해 생성된 항원-항체 반응물을 발색기질 용액을 이용하여 검출하는 단계; 및 상기 검출량이 정상인 개체에 비해 증가된 피검체를 파킨슨병에 걸렸거나 걸릴 가능성이 높은 것으로 판정하는 단계를 포함할 수 있다.In a method for detecting autoantibodies to provide Parkinson's disease diagnostic information according to another aspect of the present invention, an antigen-antibody reaction is performed by adding a biological sample isolated from a subject to a fixed body coated with cysteine protease ATG4B (ATG4B) protein. making; detecting the antigen-antibody reaction produced through the above step using a chromogenic substrate solution; And it may include the step of determining that the subject has Parkinson's disease or is highly likely to have the detection amount compared to the normal subject.

상기 발색을 유도하는 기질(발색기질)은 발색반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD(ophenylenediamine) 등을 사용할 수 있다.It is preferable to use the substrate (coloring substrate) inducing the color development according to the marker for the color reaction, TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (ophenylenediamine), and the like can be used.

보편적으로 화학발광법 (Chemiluminescence)이 주로 사용되며 이때, Luminol과 같은 발색기질은 2차 항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 3-aminophthalate를 생성하고 그 발광체의 정도를 425nm 파장의 빛으로 확인함으로써 ATG4B 단백질의 자가항체 존재 유무를 검출한다.Chemiluminescence is generally used, and at this time, a chromogenic substrate such as Luminol is decomposed by HRP used as a label of the secondary antibody conjugate to generate 3-aminophthalate, and the degree of the luminescence is measured with light with a wavelength of 425 nm. By confirming, the presence or absence of autoantibody of ATG4B protein is detected.

상기 고정체는 니트로셀룰로오스 막, PVDF 막, 폴리비닐 또는 폴리스티렌 수지로 합성된 웰 플레이트, 및 유리로 된 슬라이드 글라스로 이루어지는 군으로부터 선택될 수 있다.The fixture may be selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass.

이하에서는 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실험예 1 : 단백질칩을 이용한 마이크로 어레이 (1차 스크리닝)Experimental Example 1: Microarray using a protein chip (primary screening)

유전자 전사 조절인자들을 포함하는 약 6000 여종의 유전자 확보 후 GST tag이 부착된 재조합단백질을 유리 플레이트에 심어 단백질칩을 제작하였다. 그 후 정상군, Early stage 환자, Advanced stage 환자 중 각 두 명씩의 혈청을 1:2000의 비율로 PBST buffer (PBS, 0.5% tween 20, pH 7.4)에 추가 후 단백질칩과 실온에서 2시간 동안 반응하여 항원과 결합하도록 하였다.After securing about 6000 genes including gene transcription regulators, recombinant proteins with GST tags were planted on a glass plate to prepare a protein chip. After that, serum from two of each of the normal group, early stage patient, and advanced stage patient was added to PBST buffer (PBS, 0.5% tween 20, pH 7.4) at a ratio of 1:2000 and reacted with the protein chip at room temperature for 2 hours. to bind to the antigen.

이후 PBST를 이용하여 10분간 3번의 세척 과정을 진행한 후 1 : 5000 의 비율로 hIgG-488 (녹색 형광) 이차 항체 (Invitrogen, A-10530)를 반응시켜, 환자 혈청 내 autoantibody 존재를 확인하였다. 혈청 내 자가항체 반응 정도를 정량화 하기 위해 심겨진 단백질의 GST tag을 이용하였다. GST 항체 (Santa cruz, sc-33613)를 실온에서 2시간 동안 반응하고 동일한 wash 과정 후 rlgG-594 (적색 형광)의 이차항체 (Invitrogen, A-32740)을 통해 단백질 칩에 심겨진 단백질량을 측정하였다.After washing three times for 10 minutes using PBST, the hIgG-488 (green fluorescence) secondary antibody (Invitrogen, A-10530) was reacted at a ratio of 1:5000 to confirm the presence of autoantibody in the patient's serum. To quantify the degree of autoantibody reaction in serum, the GST tag of the implanted protein was used. The GST antibody (Santa cruz, sc-33613) was reacted at room temperature for 2 hours, and after the same wash process, the amount of protein implanted in the protein chip was measured using a secondary antibody (Invitrogen, A-32740) of rlgG-594 (red fluorescence). .

이 때 녹색 및 적색 형광을 통한 단백질 칩의 항원 항체 반응 측정에는 모두 Microarray 리더기가 사용되었다.In this case, a microarray reader was used to measure the antigen-antibody reaction of the protein chip through green and red fluorescence.

1차 스크리닝의 환자정보는 하기 표 1과 같다.The patient information of the primary screening is shown in Table 1 below.

Figure PCTKR2021001132-appb-T000001
Figure PCTKR2021001132-appb-T000001

실험예Experimental example 2 : 2 : 재조합단백질recombinant protein 정제 refine

재조합단백질 정제 벡터(pDEST15)를 BL21 대장균주로 이동(transformation)시켰다. 벡터를 포함한 BL21 대장균주 100ml을 Optical density(O.D.)가 0.6에 도달하도록 37℃ shaker에서 키운 뒤 최종 농도가 0.4 mM이 되도록 IPTG를 넣어주었다. 37℃에서 3시간의 induction 이후 BL21을 수확하여, Cell lytic B buffer (0.5% cell lytic B, 0.1% TritonX-100, 0.07% β-ME, 1X Dnase1, protease inhibitor, lysozyme in PBST)로 상온에서 10분간 반응시켜 lysis 하였다. 이 후 GST 표지된 재조합 단백질을 GST 항체가 부착된 bead 100 μl (GE, 17075601)를 이용하여, 4℃에서 3시간 동안 반응 후 원하는 단백질을pull-down 하였다.The recombinant protein purification vector (pDEST15) was transformed into the BL21 E. coli strain. 100ml of the BL21 E. coli strain containing the vector was grown on a shaker at 37°C so that the optical density (O.D.) reached 0.6, and then IPTG was added so that the final concentration was 0.4 mM. After 3 hours of induction at 37°C, BL21 was harvested, and cell lytic B buffer (0.5% cell lytic B, 0.1% TritonX-100, 0.07% β-ME, 1X Dnase1, protease inhibitor, lysozyme in PBST) was used at room temperature for 10 It was lysed by reacting for minutes. Thereafter, the GST-labeled recombinant protein was reacted with 100 μl of GST antibody-attached beads (GE, 17075601) at 4° C. for 3 hours, followed by pull-down of the desired protein.

Chromatography 칼럼 (Biorad, 731-1550)과 원심분리기를 이용하여 200g, 4℃, 5분으로 GST bead를 수확하고, Wash buffer 1 (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol, 0.1% Triton X-100, 1 mM PMSF, 0.07% tor, 로 3번 세척 후 Wash buffer 2 (50 mM Hepes pH 7.5, 100 mM NaCl, 1 mM EGTA, 10% glycerol)로 3번 세척을 진행하였다.Using a chromatography column (Biorad, 731-1550) and a centrifuge, harvest GST beads at 200 g, 4°C, 5 minutes, and wash buffer 1 (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol). , 0.1% Triton X-100, 1 mM PMSF, 0.07% tor, washed 3 times, and then washed 3 times with Wash buffer 2 (50 mM Hepes pH 7.5, 100 mM NaCl, 1 mM EGTA, 10% glycerol) .

이 후 원하는 단백질만을 추출하기 위하여, 100 μl Elution buffer (50 mM HEPES, 100mM NaCl, 30% glycerol, 40mM reduced glutathione, 0.03% triton X-100)을 활용 bead와 4℃ 1시간 반응을 통해 3번 탈착 후 Protein concentrator (Amicon, UFC5003)를 통해 정제하였다. Coomassie staining 기법을 이용하여 정제해 낸 양을 측정하였고, 정제된 단백질의 양은 BSA standard를 기준하여 판별하였다.After that, in order to extract only the desired protein, 100 μl Elution buffer (50 mM HEPES, 100mM NaCl, 30% glycerol, 40mM reduced glutathione, 0.03% triton X-100) was used and desorbed 3 times through 1 hour reaction at 4°C with beads. Then, it was purified through a protein concentrator (Amicon, UFC5003). The amount of purified protein was measured using Coomassie staining technique, and the amount of purified protein was determined based on the BSA standard.

실험예Experimental example 3 : 파일럿 스크리닝 3: Pilot screening

기 확보된 27개의 클론을 대상으로 Gateway cloning 기법을 사용하여 단백질 정제 벡터를 제작하였다. 27개의 Entry clone은 LR clonase (Invitrogen, 11791020)를 이용하여 pDEST15 vector로 옮겨졌으며, Cell lytic B 방법을 이용하여 단백질을 정제하였다.A protein purification vector was prepared using the Gateway cloning technique for 27 previously secured clones. The 27 entry clones were transferred to the pDEST15 vector using LR clonase (Invitrogen, 11791020), and the protein was purified using the Cell lytic B method.

Dot-blot 기법을 활용하여 0.1 μg 동량의 단백질을 나이트로셀룰로즈막에 도포한 뒤, 환자 혈청은 1 : 2000의 비율로 2시간 동안 상온에서 반응하여 혈청 내 자가항체가 항원 단백질과 결합시켰다.After applying 0.1 μg of protein to the nitrocellulose membrane using the dot-blot technique, the patient's serum was reacted at room temperature at a ratio of 1:2000 for 2 hours so that the autoantibody in the serum was bound to the antigen protein.

이 후 secondary hIgG-HRP를 부착한 뒤 HRP 반응을 통해 나타난 Chemiluminescence intensity를 통하여 자가 항원 항체 반응을 수치화 하였다. 전체 실험의 정규화를 위하여 양성대조군 및 항원 단백질 양이 사용되었는데, i) 양성 대조군으로는 hlgG를 동량으로 도포하여 그 양을 정량화하였고, ii) 도포된 단백질량은 GST tag 및 항체 반응으로 확인하였으며, 이 값은 수치를 정량화 할 때 사용하였다.After attaching secondary hIgG-HRP, the autoantigen antibody response was quantified through the chemiluminescence intensity shown through the HRP reaction. For normalization of the entire experiment, a positive control group and antigen protein amount were used. i) As a positive control, hlgG was applied in the same amount to quantify the amount, ii) the amount of the applied protein was confirmed by GST tag and antibody reaction, This value was used to quantify the value.

파일럿 스크리닝의 환자정보는 하기 표 2와 같다.The patient information of the pilot screening is shown in Table 2 below.

환자patient 번호number 성별gender 나이age 정상normal 1One 남male 4040 22 남male 4343 33 남male 6262 44 남male 5454 55 남male 5959 1One 여female 4848 22 여female 5555 33 여female 5858 44 여female 4949 55 여female 4646 파킨슨병Parkinson's disease 1One 남male 4848 22 남male 4343 33 남male 6060 44 남male 6464 55 남male 4646 1One 여female 5151 22 여female 4545 33 여female 4949 44 여female 4848 55 여female 4444

실험예Experimental example 4 : 2차4: 2nd 스크리닝 screening

1차 단백질 칩 마이크로 어레이 및 파일럿 스크리닝을 대상으로 환자군에서 유의미한 변화를 도출했던 ATG4B 단백질의 자가항체를 30명 이상의 실험군에서 스크리닝을 진행하였다.Autoantibodies of ATG4B protein, which derived significant changes in the patient group, were screened in more than 30 experimental groups for the primary protein chip microarray and pilot screening.

동량의 GST 단백질과, GST-ATG4B 단백질을 나이트로셀룰로즈막에 도포하고, 정상군 및 환자군 혈청을 1:2000의 비율로 2시간 동안 상온에서 반응시키고, 이후 hIgG-HRP를 이용한 Chemiluminescence 반응으로, 그 결과를 확인하였으며, 동량의 GST, 및 GST-ATG4B의 도포는 GST 안티바디를 사용하여 확인하였다.The same amount of GST protein and GST-ATG4B protein was applied to the nitrocellulose membrane, and the serum of the normal group and the patient group was reacted at room temperature at a ratio of 1:2000 for 2 hours, followed by a Chemiluminescence reaction using hIgG-HRP. The results were confirmed, and the application of the same amount of GST and GST-ATG4B was confirmed using the GST antibody.

총 31명의 정상군과 42명의 환자군이 사용되었으며, 개인별 ATG4B 자가항체 보유량이 측정되었고, GST 단백질을 음성대조군으로 사용하여, 불필요한 시그널이 나오는 것을 방지하고, 정확한 결과를 확인할 수 있도록 하였다.A total of 31 normal and 42 patient groups were used, the amount of ATG4B autoantibody per person was measured, and the GST protein was used as a negative control to prevent unnecessary signals from appearing and to confirm accurate results.

2차 스크리닝의 환자정보는 하기 표 3과 같다.The patient information of the secondary screening is shown in Table 3 below.

Figure PCTKR2021001132-appb-T000002
Figure PCTKR2021001132-appb-T000002

Figure PCTKR2021001132-appb-I000001
Figure PCTKR2021001132-appb-I000001

Figure PCTKR2021001132-appb-I000002
Figure PCTKR2021001132-appb-I000002

실시예 1 : 단백질칩을 이용한 마이크로 어레이 사용 1차 스크리닝 결과Example 1: Results of primary screening using a microarray using a protein chip

정상군 및 Early stage 와 Advanced stage PD(파킨슨병) 환자군을 각 2명씩 선정하여, 6000개 단백질이 심겨진 protein chip과 반응시켰다.Normal group and early stage and advanced stage PD (Parkinson's disease) patients were selected for each group and reacted with a protein chip implanted with 6,000 proteins.

도 1은 6000개 단백질 칩을 이용한 파킨슨병 환자 및 정상군 Autoantibody 반응 방법을 나타낸 것이다1 shows the autoantibody reaction method of Parkinson's disease patients and normal groups using 6000 protein chips.

그 결과, Early stage PD 환자에서만 변하는 단백질을 포함하여 총 44개의 단백질이 환자군에서 변하는 것을 확인할 수 있었다.As a result, it was confirmed that a total of 44 proteins were changed in the patient group, including those that were changed only in early stage PD patients.

첫 번째로 환자군에서 증가하는 18개 단백질은, RAD23B, VAMP4, VAMP3, PKNOX1, CSAG2, VAMP2, C6orf106, MUTYH, PMF1, LARP7, SNPR70, RAB10, C14orf106, TIMM44, ZNF503, HSF1, ATF6, POLR1A 로 확인되었고, 대부분의 경우 Early stage보다 Advanced PD환자군에서 증가한 것을 확인할 수 있었다 (도 2).First, 18 proteins increasing in the patient group were identified as RAD23B, VAMP4, VAMP3, PKNOX1, CSAG2, VAMP2, C6orf106, MUTYH, PMF1, LARP7, SNPR70, RAB10, C14orf106, TIMM44, ZNF503, HSF1, ATF6, POLR1A. , it was confirmed that, in most cases, it was increased in the advanced PD patient group than in the early stage (Fig. 2).

환자군에서 감소한 것으로 드러난 단백질은 14개로, STK24, NDUFV2, FLJ37087, ANXA2, LACTTB2, CLIC1, MSRA, YWHAE, ACOX1, G6PD, MRPS36, ATP5L, HSPA8, ZNF516 로 나타났으며, 두 환자군에서 모두 감소하는 경향으로 확인되었다 (도 3).The 14 proteins found to be decreased in the patient group were STK24, NDUFV2, FLJ37087, ANXA2, LACTTB2, CLIC1, MSRA, YWHAE, ACOX1, G6PD, MRPS36, ATP5L, HSPA8, and ZNF516. was confirmed (Fig. 3).

세번째로 12개의 단백질은 Early stage와 Advanced stage PD 환자에서 서로 다른 양상이 나타났다.Third, 12 proteins showed different patterns in early stage and advanced stage PD patients.

Early stage에서 그 양이 감소하였으나 Advanced stage에서 증가하는 단백질은, SF3B4, SOD1, MPP1, DHODH, PECI, ZYX, ATG4B, OGG1, CBX1, GRHPR, FHL3 로 나타났다. SMARCA1의 경우는 Early stage에서 그 양이 증가하였으나, Advanced PD환자에서 감소하는 패턴을 나타냈다 (도 4). Proteins that decreased in the early stage but increased in the advanced stage were SF3B4, SOD1, MPP1, DHODH, PECI, ZYX, ATG4B, OGG1, CBX1, GRHPR, and FHL3. In the case of SMARCA1, the amount increased in the early stage, but showed a decreasing pattern in the advanced PD patients (Fig. 4).

실시예 2 : 파일럿 스크리닝 결과Example 2: Pilot Screening Results

1차 Microarray 스크리닝을 대상으로 확인한 44개 단백질 중 기 확보된 27개의 단백질을 단백질 정제 벡터로 옮겨 그 단백질을 추출하였고, 이 후 GST 단백질을 음성대조군으로, hIgG를 양성대조군으로 확립하여, 27개 단백질을 활용한 Mini-chip을 제작하였다 (도 5 및 도 6). 각 20개의 Mini-chip을 제작하여, 정상군 및 환자군 각 10명을 대상으로 그 결과를 확인하였다.Among the 44 proteins identified in the primary microarray screening, 27 proteins were transferred to a protein purification vector to extract the proteins. After that, GST protein was established as a negative control group and hIgG as a positive control group, and 27 proteins were established. A Mini-chip was manufactured using ( FIGS. 5 and 6 ). Each of 20 mini-chips was manufactured, and the results were checked for each of 10 patients in the normal group and the patient group.

이 때 환자군의 성별에 의한 변화를 확인하기 위하여, 남, 여 각 5명씩을 대상으로 한 결과를 비교하였다.At this time, in order to confirm the change according to the gender of the patient group, the results of 5 males and 5 females each were compared.

이 때, 환자군에서 증가하는 단백질은 8 개로, PECI, ATG4B, RAB10, ATP5L, GRHRP, LACTTB2, G6PD, OGG1으로 나타났으며, 감소하는 19개 단백질은 MPP1, C6orf106, FHL3, SNRP70, MRPS36, ATF6, RAD23B, VAMP2, ANXA2, ACOX1, ZNF503, HSPA8, LARP7, NDUFV2, CLIC1, CSAG2, VAMP3, YHWAE, STK24 이며 성별에 따른 뚜렷한 차이는 보이지 않았다.At this time, 8 proteins increased in the patient group, PECI, ATG4B, RAB10, ATP5L, GRHRP, LACTTB2, G6PD, OGG1, and 19 proteins that decreased were MPP1, C6orf106, FHL3, SNRP70, MRPS36, ATF6, RAD23B, VAMP2, ANXA2, ACOX1, ZNF503, HSPA8, LARP7, NDUFV2, CLIC1, CSAG2, VAMP3, YHWAE, STK24, and there was no significant difference according to gender.

파일럿 스크리닝 결과, ATG4B 단백질이 환자군에서 유의미한 증가한 것을 확인할 수 있었고, ATG4B 단백질은 1차 스크리닝에서도 환자군에서 증가하는 것으로 나타난 단백질로써, 환자군에서 공통적으로 증가하는 양상을 보였다 (도 7).As a result of the pilot screening, it was confirmed that the ATG4B protein significantly increased in the patient group, and the ATG4B protein was found to be increased in the patient group even in the primary screening, and showed a common increase in the patient group ( FIG. 7 ).

ATG4B 단백질의 경우, 세포의 Autophagy에 관여하는 단백질로써, 파킨슨병을 비롯한 많은 퇴행성 뇌질환에서 그 기능이 변화되어 있는 것으로 알려져 있고, 본 연구진은 1차 및 파일럿 스크리닝을 거쳐 발굴된 ATG4B 단백질을 중심으로 30명 이상의 환자군에 검증 절차를 진행하였다.In the case of ATG4B protein, as a protein involved in cellular autophagy, its function is known to be changed in many degenerative brain diseases including Parkinson's disease. The validation procedure was performed on more than 30 patient groups.

실시예 3 : 2차 스크리닝 결과Example 3: Second screening results

ATG4B 단백질을 대상으로 정상군 31명, 환자군 42명을 대상으로 2차 스크리닝을 진행하였다. 상기 2차 스크리닝 실험은 Blind로 진행하여 실험자의 개입을 최소한으로 하였다.Secondary screening was performed for ATG4B protein in 31 normal subjects and 42 patients in the patient group. The second screening experiment was performed blindly to minimize the intervention of the experimenter.

또한, 실험 이후 환자 정보 확인을 통해 ATG4B에 반응하는 다양한 요인을 확인하였다.In addition, various factors responding to ATG4B were identified through the confirmation of patient information after the experiment.

2차 스크리닝 결과 90% 이상의 PD 환자에서 높은 ATG4B autoantibody의 양이 존재하는 것을 확인할 수 있었으며, 성별 및 나이 분포에서도 모두 고르게 높은 반응을 나타내었다 (도 8).As a result of the secondary screening, it was confirmed that a high amount of ATG4B autoantibody was present in more than 90% of PD patients, and both gender and age distribution showed a high response evenly ( FIG. 8 ).

도 8에 나타낸 바와 같이 ATG4B 씨그널을 cut-off value로 설정시 정상군에서 31명 중 2명이 양성으로 검출되었고, 파킨슨 환자군에서 42명 중 38명이 양성으로 검출되었다. 따라서, 정상군에 대한 특이도 93.5%, 민감도 90.5%로 측정되었다 (표 4, 정상군과 환자군에서의 자가항체 테스트 결과).As shown in FIG. 8 , when the ATG4B signal was set as a cut-off value, 2 out of 31 patients in the normal group were detected as positive, and 38 out of 42 in the Parkinson's patient group were detected as positive. Therefore, it was measured to have a specificity of 93.5% and a sensitivity of 90.5% for the normal group (Table 4, autoantibody test results in the normal group and the patient group).

질병 양성disease benign 질병 음성disease negative 전체entire 테스트 양성test positive 3838 22 4040 테스트 음성test voice 44 2929 3333 전체entire 4242 3131 7373

본 연구를 통하여, Early 및 Advanced PD 환자 특이적으로 변하는 Autoantibody를 확인할 수 있었으며, 환자의 질병 진행 정보 및 환자정보를 바탕으로 한 연구를 통해 환자 맞춤 진단을 가능케 할 것으로 사료된다.Through this study, it was possible to identify autoantibodies that change specifically for patients with Early and Advanced PD, and it is thought that a patient-specific diagnosis will be possible through a study based on the patient's disease progression information and patient information.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하기 위하여, 상기 자가항체에 특이적으로 결합하는 ATG4B 단백질을 포함하는 파킨슨병 진단용 조성물.A composition for diagnosing Parkinson's disease, comprising the ATG4B protein specifically binding to the autoantibody in order to measure the level of the autoantibody against the cysteine protease ATG4B (ATG4B) protein. 제1항에 있어서, According to claim 1, 상기 ATG4B 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함하는 파킨슨병 진단용 조성물.The ATG4B protein is a composition for diagnosing Parkinson's disease comprising the amino acid sequence represented by SEQ ID NO: 1. 제1항 또는 제2항의 조성물을 포함하는 파킨슨병 진단용 키트.A kit for diagnosing Parkinson's disease comprising the composition of claim 1 or 2. 제3항에 있어서,4. The method of claim 3, 상기 키트는 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체(autoantibody)를 검출하는 파킨슨병 진단용 키트.The kit is a kit for diagnosing Parkinson's disease for detecting an autoantibody to the ATG4B (cysteine protease ATG4B) protein. 제4항에 있어서,5. The method of claim 4, 상기 자가항체의 검출은 ELISA(Enzyme-linked immunosorbent assay), RIA(Radioimmnoassay), 샌드위치 측정법(Sandwich assay), 웨스턴 블롯(Western Blot), 면역블롯 분석(Immunoblot assay), 및 면역조직화학염색 방법(Immnohistochemical staining)으로 이루어지는 군으로부터 선택되는 하나 이상으로 수행되는 파킨슨병 진단용 키트.The detection of the autoantibody is ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmnoassay), sandwich assay (Sandwich assay), Western blot (Western Blot), immunoblot analysis (Immunoblot assay), and immunohistochemical method (Immnohistochemical) A kit for diagnosing Parkinson's disease performed with one or more selected from the group consisting of staining). 피검체로부터 분리된 생물학적 시료로부터 ATG4B(cysteine protease ATG4B) 단백질에 대한 자가항체의 수준을 측정하는 단계;measuring the level of an autoantibody to the cysteine protease ATG4B (ATG4B) protein from the biological sample isolated from the subject; 상기 측정된 자가항체 수준을 정상인 개체로부터 분리된 생물학적 시료로부터 측정된 ATG4B 단백질에 대한 자가항체 수준과 비교하는 단계: 및comparing the measured autoantibody level with the autoantibody level to the ATG4B protein measured from a biological sample isolated from a normal subject; and 상기 피검체의 자가항체 수준이 상기 정상인 개체의 자가항체 수준보다 높은 경우 파킨슨병 양성으로 판정하는 단계;determining that the subject's autoantibody level is higher than that of the normal subject as positive for Parkinson's disease; 를 포함하는 파킨슨병 진단을 위한 정보제공 방법.Information providing method for diagnosing Parkinson's disease, including. 제 6 항에 있어서,7. The method of claim 6, 상기 생물학적 시료는 혈액, 혈장, 및 혈청으로 이루어진 군으로부터 선택되는 파킨슨병 진단을 위한 정보제공 방법.The biological sample is an information providing method for diagnosing Parkinson's disease selected from the group consisting of blood, plasma, and serum. 제6항에 있어서,7. The method of claim 6, 상기 ATG4B 단백질에 대한 자가항체 수준은 상기 자가항체와의 항원-항체 반응을 이용하여 측정되는 파킨슨병 진단을 위한 정보제공 방법.An information providing method for diagnosing Parkinson's disease, wherein the autoantibody level against the ATG4B protein is measured using an antigen-antibody reaction with the autoantibody. 제6항에 있어서, 7. The method of claim 6, 상기 ATG4B 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함하는 파킨슨병 진단을 위한 정보제공 방법.The ATG4B protein is an information providing method for diagnosing Parkinson's disease comprising the amino acid sequence represented by SEQ ID NO: 1. ATG4B(cysteine protease ATG4B) 단백질이 코팅된 고정체에 피검체로부터 분리된 생물학적 시료를 가하여 항원-항체 반응시키는 단계;A method comprising: adding a biological sample isolated from a subject to a cysteine protease ATG4B (ATG4B) protein-coated fixture, followed by antigen-antibody reaction; 상기 단계를 통해 생성된 항원-항체 반응물을 발색기질 용액을 이용하여 검출하는 단계; 및detecting the antigen-antibody reaction produced through the above step using a chromogenic substrate solution; and 상기 검출량이 정상인 개체에 비해 증가된 피검체를 파킨슨병에 걸렸거나 걸릴 가능성이 높은 것으로 판정하는 단계;determining that the subject has or is highly likely to have Parkinson's disease, compared to the normal subject; 를 포함하는 파킨슨병 진단 정보를 제공하기 위해 자가 항체를 검출하는 방법.A method of detecting autoantibodies to provide diagnostic information for Parkinson's disease comprising a. 제10항에 있어서,11. The method of claim 10, 상기 고정체는 니트로셀룰로오스 막, PVDF 막, 폴리비닐 또는 폴리스티렌 수지로 합성된 웰 플레이트, 및 유리로 된 슬라이드 글라스로 이루어지는 군으로부터 선택되는 파킨슨병 진단 정보를 제공하기 위해 자가 항체를 검출하는 방법.The method for detecting autoantibodies to provide diagnostic information for Parkinson's disease, wherein the fixture is selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass. 제10항에 있어서,11. The method of claim 10, 상기 ATG4B 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함하는 파킨슨병 진단 정보를 제공하기 위해 자가 항체를 검출하는 방법.The ATG4B protein is a method of detecting an autoantibody to provide Parkinson's disease diagnostic information comprising the amino acid sequence represented by SEQ ID NO: 1.
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