Novel antibody, as well as method, kit, apparatus, and computer program for measuring acetylated α-synuclein, information acquisition method, and diagnostic biomarker

Abstract

To provide a technology capable of detecting acetylated α-synuclein.SOLUTION: An antibody of the present disclosure is an antibody that recognizes an acetylated epitope including an acetylated amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. A method for measuring acetylated α-synuclein according to the present disclosure includes a reaction step of allowing the antibody of the present disclosure to bind to acetylated α-synuclein in a biological sample collected from a subject, and a detection step of detecting the acetylated α-synuclein bound to the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel antibody, a method, a kit, an apparatus and a computer program for measuring acetylated alpha-synuclein, an information obtaining method, and a diagnostic biomarker. [Background technology]

[0002] Parkinson's disease (PD) and atypical parkinsonian syndromes (particularly multiple system atrophy (MSA)) have different brain pathologies, and current antiparkinsonian drugs are ineffective against MSA. Therefore, the prognoses for the two conditions are significantly different. Differentiating between PD and MSA, both of which occur frequently in elderly people, is important for clinical practice in neurology and geriatrics. However, it is difficult to distinguish between PD and MSA based solely on the patient's clinical symptoms and imaging tests such as MRI, especially in the early stages of the disease. Therefore, objective body fluid biomarkers (particularly blood biomarkers) are needed, and various research and development efforts are underway.

[0003] Non-Patent Document 1 discloses that α-synuclein (α-syn) in cerebrospinal fluid is useful as a diagnostic biomarker for PD. Non-Patent Document 2 discloses a method for measuring α-syn oligomers in cerebrospinal fluid, which has high sensitivity and specificity for diagnosing PD.

[0004] Non-Patent Documents 3 and 4 disclose that exosomes in the blood and the α-syn and other substances contained therein are useful for distinguishing between PD and atypical Parkinson's syndrome.

[0005] Furthermore, Non-Patent Document 5 discloses that it is possible to differentiate between PD and MSA by amplifying highly aggregating α-syn in cerebrospinal fluid using a method known as RT-QuIC. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tokuda T, et al. Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson's disease. Biochem Biophys Res Commun. 2006; 349(1): 162-6. [Non-patent document 2] Tokuda T, et al. Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease. Neurology. 2010;75(20):1766-1772. [Non-patent document 3] Ohmichi T, Tokuda T, et al. Quantification of brain-derived extracellular vesicles in plasma as a biomarker to diagnose Parkinson's and related diseases. Parkinsonism Relat Disord. 2019; 61: 82-87. [Non-patent document 4] Shahnawaz M, et al. Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy. Nature. 2020; 578(7794): 273-277. [Non-patent document 5] Jiang C, et al. Validation of α-Synuclein in L1CAM-Immunocaptured Exosomes as a Biomarker for the Stratification of Parkinsonian Syndromes. Mov Disord. 2021; 36(11): 2663-2669. Summary of the Invention [Problem to be solved by the invention]

[0007] The techniques disclosed in Non-Patent Documents 1 and 2 cannot be applied to detecting α-syn in blood. The techniques disclosed in Non-Patent Documents 3 and 4 have not been fully verified and have not yet been put to practical use. The technique disclosed in Non-Patent Document 5 is a qualitative method, not a quantitative method. Furthermore, the technique disclosed in Non-Patent Document 5 has not yet been applied to detecting α-syn in blood. In other words, a blood biomarker that can distinguish between PD and atypical parkinsonism (particularly MSA) has not yet been realized, and the development of such a blood biomarker is desired.

[0008] One aspect of the present invention is to provide a technique that can detect acetylated α-syn. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention includes the following aspects. An antibody that recognizes an acetylated epitope containing an acetylated amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. A method for obtaining information on whether a subject suffers from Parkinson's disease or atypical parkinsonism, comprising the step of measuring the concentration of acetylated alpha-synuclein in a biological sample collected from the subject. Diagnostic biomarkers for Parkinson's disease or atypical parkinsonism, including acetylated alpha-synuclein. [Effects of the Invention]

[0010] According to one embodiment of the present invention, acetylated α-syn can be detected. [Brief explanation of the drawings]

[0011] [Figure 1]1 is a diagram showing a configuration of a main part of a measurement device according to an embodiment of the present invention; [Figure 2] 1 is a diagram illustrating a configuration of a main part of an information acquisition device according to an aspect of the present invention. [Figure 3] FIG. 1 shows the evaluation results of SA17E40 antibody. [Figure 4] FIG. 1 shows the evaluation results of the SB20F51 antibody. [Figure 5] FIG. 1 shows the evaluation results of the SB20F56 antibody. [Figure 6] FIG. 10 shows the results of measuring Ac-α-syn concentration by a measurement method using SA17E40 antibody in Evaluation Example 2. [Figure 7] FIG. 10 shows the results of measuring Ac-α-syn concentration by a measurement method using the SB20F51 antibody in Evaluation Example 2. [Figure 8] FIG. 10 shows the results of measuring Ac-α-syn concentration by a measurement method using the SB20F56 antibody in Evaluation Example 2. [Figure 9] FIG. 1 shows the evaluation results of S08H8 antibody. [Figure 10] FIG. 1 shows the evaluation results of the S08H17 antibody. [Figure 11] FIG. 10 shows the results of measuring Ac-α-syn concentrations by a measurement method using S08H8 antibody in Evaluation Example 4. [Figure 12] FIG. 10 shows the results of measuring Ac-α-syn concentrations by a measurement method using the S08H17 antibody in Evaluation Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0012] [New antibody] An antibody according to one embodiment of the present invention is an antibody that recognizes an acetylated epitope containing an acetylated amino acid residue in the amino acid sequence of α-synuclein (hereinafter, sometimes referred to as an "anti-Ac-α-syn antibody").

[0013] An example of α-synuclein is the human-derived amino acid sequence shown in SEQ ID NO: 1 (UniProtKB accession No: P37840.1).

[0014] As used herein, the term "acetylated amino acid residue" refers to an amino acid residue to which an acetyl group has been added. In α-synuclein, examples of amino acid residues that can be acetylated include the methionine residue at position 1 in the amino acid sequence of SEQ ID NO: 1, and the lysine residues at positions 6, 10, 12, 21, 23, 32, 34, 43, 45, 58, 60, 80, 96, 97, and 102.

[0015] The acetylated epitope comprises an acetylated methionine residue at position 1 of the amino acid sequence of SEQ ID NO: 1, and / or at least one acetylated lysine residue selected from the group consisting of positions 6, 10, 12, 21, 23, 32, 34, 43, 45, 58, 60, 80, 96, 97, and 102.

[0016] The number of amino acids constituting the acetylated epitope is not limited, and may be, for example, 2 to 60, preferably 3 to 45, more preferably 4 to 30, and even more preferably 5 to 20.

[0017] The acetylated epitope may be an epitope containing an acetylated amino acid residue in the amino acid sequence at positions 1 to 60, 1 to 50, or 1 to 40 of the amino acid sequence shown in SEQ ID NO:1.

[0018] Preferably, an antibody according to one embodiment of the present invention does not recognize an epitope that does not contain acetylated amino acid residues in the amino acid sequence of α-synuclein.

[0019] The antibody according to one embodiment of the present invention may be any of polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab)2, etc.). The immunoglobulin class and subclass are not particularly limited. Antibodies selected from antibody libraries by phage display or other methods may also be used.

[0020] Furthermore, the antibody according to one aspect of the present invention may be a non-human antibody derived from a mouse, rat, rabbit, goat, or the like, a chimeric antibody, a humanized antibody, or a human antibody.

[0021] Furthermore, the antibody according to one aspect of the present invention may be labeled with a fluorescent dye, an enzyme, a radioactive label, or the like.

[0022] The antibody according to one embodiment of the present invention can be produced by means known to those skilled in the art. For example, the antibody can be produced by immunizing a mammal (e.g., mouse, rat, rabbit, etc.) with α-synuclein or a fragment thereof having an acetylated amino acid residue. Alternatively, the antibody can be produced using recombinant DNA techniques.

[0023] [Method for measuring acetylated α-synuclein] A measurement method according to one embodiment of the present invention is a method for measuring acetylated α-synuclein, which comprises a reaction step and a detection step.

[0024] As used herein, acetylated α-synuclein refers to α-synuclein having at least one acetylated amino acid residue in its amino acid sequence. Examples of amino acid residues that can be acetylated are as described above.

[0025] (Reaction step) In the reaction step, the anti-Ac-α-syn antibody is bound to acetylated α-synuclein in a biological sample collected from a subject.

[0026] Examples of subjects include mammals, birds, reptiles, amphibians, etc., with mammals being preferred. Mammals include humans and non-human animals. Non-human animals include livestock such as cows, horses, pigs, and sheep, as well as pets or laboratory animals such as dogs, cats, rats, mice, hamsters, monkeys, and rabbits. Humans are preferred. Birds include poultry such as chickens, ducks, and geese.

[0027] The biological sample collected from a subject is not particularly limited as long as it can contain acetylated α-synuclein. Examples of biological samples include blood samples, saliva, cerebrospinal fluid (CSF), urine, and tissue extract samples. Examples of tissue extracts include fluids extracted from tissues such as the brain, peripheral nerves, muscles, spinal cord, sympathetic ganglia, skin, digestive tract, heart, and red blood cells.

[0028] The biological sample is preferably a blood sample because it is minimally invasive and allows for easy measurement. Blood samples include whole blood, plasma, serum, extracellular vesicles, etc., and the blood sample is preferably plasma or serum, more preferably plasma. Plasma can be separated from whole blood collected as a heparin salt (preferably sodium salt), citrate salt (preferably sodium salt), or ethylenediaminetetraacetic acid (EDTA) salt (preferably sodium salt or potassium salt). More preferably, the plasma is plasma separated from whole blood collected as an EDTA salt or heparin salt. Extracellular vesicles can be separated from whole blood by ultracentrifugation, polymer precipitation, chromatography, or the like.

[0029] (Detection process) In the detection step, acetylated α-synuclein bound to the anti-Ac-α-syn antibody is detected. For example, acetylated α-synuclein bound to the antibody can be detected by immunoassay or immunoprecipitation-mass spectrometry. Immunoprecipitation-mass spectrometry (IP-MS) is a measurement method that combines immunoprecipitation and mass spectrometry.

[0030] The measurement principle of the immunoassay may be a sandwich method, a direct adsorption method, a competitive method, etc. The sandwich method is preferred in terms of high detection sensitivity and simple operation.

[0031] Examples of the immunoassay include enzyme immunoassay (EIA), enzyme-linked immunosorbent (ELISA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay (FIA), electrochemiluminescent immunoassay (ECLIA), bioluminescent immunoassay (BLIA), immunochromatography, immunoturbidimetry (TIA), Western blotting, immunohistological staining, flow cytometry, immunoprecipitation, and latex agglutination turbidimetry (LA). In terms of high detection sensitivity and simple operation, the immunoassay is preferably ELISA, CLEIA, CLIA, or ECLIA, and more preferably ELISA. Examples of ELISA include sandwich ELISA, direct adsorption ELISA, and competitive ELISA. Among ELISAs, sandwich ELISA is more preferred due to its high detection sensitivity and simple operation. The immunoassay can be performed according to procedures known to those skilled in the art.

[0032] When a sandwich-type ELISA is used as the immunoassay, an anti-Ac-α-syn antibody can be used as the first antibody, and an antibody that recognizes α-synuclein and recognizes an epitope different from the acetylated epitope recognized by the anti-Ac-α-syn antibody can be used as the second antibody. When a sandwich-type immunoassay is used as the immunoassay, one of the first and second antibodies can be used as a capture antibody (antibody immobilized on a solid phase), and the other antibody can be used as a detection antibody (antibody carrying a labeled substance). When two or more types of anti-Ac-α-syn antibodies are used as the first antibody, it is preferable to use the first antibody as the detection antibody.

[0033] An example of the second antibody is an antibody that recognizes an epitope contained in a peptide consisting of amino acids 90 to 140 (or 90 to 120, or 90 to 100) of the amino acid sequence shown in SEQ ID NO: 1. An example of a commercially available second antibody is Syn-1 (Transduction Laboratories).

[0034] The second antibody may be a polyclonal antibody, a monoclonal antibody, or a fragment thereof (e.g., Fab, F(ab)2, etc.). The immunoglobulin class and subclass are not particularly limited. An antibody selected from an antibody library by a phage display method or the like may also be used. The second antibody may also be a commercially available antibody.

[0035] Examples of the solid phase on which the capture antibody is immobilized include a plate, a substrate, beads, particles, a membrane, etc. Examples of the material of the solid phase include silica, alumina, glass, metal, resin, nitrocellulose, etc.

[0036] The labeling substance carried by the detection antibody is not particularly limited as long as it generates a detectable signal. For example, it may be a substance that generates a signal by itself (hereinafter also referred to as a "signal-generating substance"), or a substance that generates a signal by catalyzing the reaction of another substance. Examples of signal-generating substances include fluorescent substances and radioisotopes. Examples of substances that catalyze the reaction of another substance to generate a detectable signal include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, luciferase, etc. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioisotopes include 125I, 14C, and 32P. Among these, enzymes are preferred as labeling substances, and β-galactosidase is particularly preferred.

[0037] When a sandwich immunoassay is used as the immunoassay, acetylated α-synuclein can be measured by forming an immune complex between acetylated α-synuclein, a capture antibody, and a detection antibody, and then detecting the labeled substance bound to the detection antibody.

[0038] The immune complex may be formed by forming an immune complex between acetylated α-synuclein in a biological sample, a capture antibody, and a detection antibody in a buffer solution, and then binding the immune complex to a carrier. For example, the immune complex can be immobilized on the carrier by contacting a complex of a capture antibody, acetylated α-synuclein, and a biotin-labeled detection antibody with a carrier on which avidin or streptavidin has been immobilized.

[0039] In the measurement method according to one embodiment of the present invention, a commercially available measurement device using an immunoassay may be used. Examples of devices using a sandwich-type ELISA include the Simoa™ HD-1 system and Simoa™ HD-X system manufactured by Quanterix, LUMIPULSE™ manufactured by Fujirebio Inc., SQ120MM manufactured by Meso Scale Diagnostics, ARCHITECT™ manufactured by Abbott Pharmaceuticals, and HI-1000 manufactured by Sysmex Corporation.

[0040] The Simoa™ HD-1 system and Simoa™ HD-X system manufactured by Quanterix are equipped with an imaging device in the detection section and are integrated with a device for measuring acetylated α-synuclein, which will be described later. Examples of measuring target substances in biological samples using the Simoa™ HD-1 system or Simoa™ HD-X system are shown in Japanese Patent Nos. 7450995 and 6961278, etc.

[0041] The detection results obtained in the detection step may be converted into measured values ​​of acetylated α-synuclein using a calibration curve. Here, the measured value of acetylated α-synuclein refers to a value reflecting the amount or concentration of acetylated α-synuclein. When the measured value is expressed as "amount," it may be in molar or mass, but is preferably expressed in mass. When the value is expressed as "concentration," it may be in molar concentration or the ratio of mass per a fixed volume of biological sample (mass / volume), but is preferably expressed in mass / volume.

[0042] The measurement method according to one aspect of the present invention can determine the presence or absence of acetylated α-synuclein in a biological sample and the amount of acetylated α-synuclein in the biological sample, and therefore can be used for qualitative or quantitative analysis.

[0043] [Kit for measuring acetylated α-synuclein] A kit according to one embodiment of the present invention is a kit for measuring acetylated α-synuclein, which comprises one or more anti-Ac-α-syn antibodies.

[0044] A kit according to one embodiment of the present invention may contain, in addition to an anti-Ac-α-syn antibody, an antibody recognizing an epitope different from the acetylated epitope recognized by the anti-Ac-α-syn antibody. The kit may also include reagents and instruments used in immunoassays, such as a blocking agent (e.g., albumin, casein, or skim milk), a carrier (e.g., beads), and a buffer solution (which may contain the blocking agent and EDTA) used to suspend the carrier and dilute the sample. The kit may also contain multiple different reagents mixed in appropriate volumes and / or forms, or each reagent may be provided in a separate container. The reagent kit may also include instructions describing the procedure for measuring acetylated α-synuclein. The instructions may be written or printed on paper or other media, or may be attached to electronic media such as magnetic tape, a computer-readable disk, or a CD-ROM.

[0045] Furthermore, the kit according to one embodiment of the present invention may include a computer-readable recording medium storing a computer program for measuring acetylated α-synuclein. Examples of the recording medium include a hard disk, a semiconductor memory device such as a flash memory, and an optical disk.

[0046] Another aspect of the present invention is a kit for obtaining information that aids in determining whether a person has Parkinson's disease or atypical Parkinson's syndrome, the kit including the detection reagent. The kit may also include a computer-readable recording medium storing a computer program for obtaining information that aids in determining whether a person has Parkinson's disease or atypical Parkinson's syndrome. The kit can also determine whether a person has Parkinson's disease or atypical Parkinson's syndrome.

[0047] Another aspect of the present invention is a kit for obtaining information on whether a patient has Parkinson's disease or atypical parkinsonism, comprising one or more anti-Ac-α-syn antibodies. The kit may also include a computer-readable recording medium storing a computer program for obtaining information that assists in determining whether a patient has Parkinson's disease or atypical parkinsonism. Examples of atypical parkinsonism include multiple system atrophy (MSA), progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, cerebrovascular parkinsonism, postencephalitic parkinsonism, hereditary spinocerebellar ataxia (types 1, 2, and 3), hydrocephalus, traumatic brain injury, drug-induced parkinsonism, hypoparathyroidism, and manganese poisoning.

[0048] Another aspect of the present invention is a kit for obtaining information on whether a subject is normal, has Parkinson's disease, or has atypical Parkinsonism, the kit comprising one or more anti-Ac-α-syn antibodies. The kit may also include a computer-readable recording medium storing a computer program for obtaining information that aids in determining whether a subject is normal, has Parkinson's disease, or has atypical Parkinsonism.

[0049] [Information acquisition method] An information obtaining method according to one embodiment of the present invention is a method for obtaining information on whether a subject suffers from Parkinson's disease or atypical parkinsonism, comprising the step of measuring the concentration of acetylated α-synuclein in a biological sample collected from the subject. Methods for measuring the concentration of acetylated α-synuclein include immunoassays using an antibody according to one embodiment of the present invention, measurement by high-performance liquid chromatography (HPLC), and measurement by mass spectrometry such as LC / MS.

[0050] Another aspect of the present invention also includes an information obtaining method for obtaining information on whether a subject is suffering from Parkinson's disease, atypical Parkinson's syndrome, or neither Parkinson's disease nor atypical Parkinson's syndrome, based on the amount of acetylated α-synuclein detected by a measurement method according to one aspect of the present invention.

[0051] An information acquisition method according to one embodiment of the present invention may include a comparison step of comparing the detection result (concentration or detected amount) of acetylated α-synuclein in a biological sample collected from a subject with a predetermined reference value. An example of the predetermined reference value is the detection result (concentration or detected amount) of acetylated α-synuclein in a biological sample derived from a subject with Parkinson's disease or atypical Parkinson's syndrome. When the detection result of acetylated α-synuclein in the biological sample derived from a subject with Parkinson's disease is a predetermined reference value and the detection result of acetylated α-synuclein in the biological sample collected from the subject is increased or decreased compared to the predetermined reference value, information indicating that the subject is suffering from atypical Parkinson's syndrome is acquired. More preferably, information indicating that the subject is suffering from atypical Parkinson's syndrome is acquired when the detection result of acetylated α-synuclein in the biological sample is increased above the predetermined reference value. When the detection result of acetylated α-synuclein in a biological sample derived from a subject with atypical Parkinson's syndrome is a predetermined reference value and the detection result of acetylated α-synuclein in a biological sample collected from the subject is increased or decreased from the predetermined reference value, information indicating that the subject is suffering from Parkinson's disease is obtained. More preferably, when the detection result of acetylated α-synuclein in the biological sample is increased above the predetermined reference value, information indicating that the subject is suffering from Parkinson's disease is obtained.

[0052] The predetermined reference value may be a cutoff value set by ROC (receiver operating characteristic curve) analysis.

[0053] In one embodiment of the information acquisition method of the present invention, for example, the concentration of acetylated α-synuclein in a biological sample collected from a subject is measured using two or more anti-Ac-α-syn antibodies with different epitopes, thereby obtaining information on whether the subject is normal, suffers from Parkinson's disease, suffers from atypical Parkinson's syndrome, or suffers from neither Parkinson's disease nor atypical Parkinson's syndrome.

[0054] Furthermore, the information acquisition method according to one embodiment of the present invention may be able to obtain information as to whether a subject is normal, suffers from Parkinson's disease, suffers from multiple system atrophy, or suffers from an atypical parkinsonism other than multiple system atrophy, for example, by measuring the concentration of acetylated alpha-synuclein in a biological sample collected from the subject using two or more anti-Ac-alpha-syn antibodies with different epitopes.

[0055] For example, the concentration of acetylated α-synuclein in a biological sample collected from a subject may be measured using anti-Ac-α-syn antibody A (e.g., an antibody that specifically recognizes acetylation of at least one lysine selected from lysines 12, 34, and 45 of SEQ ID NO: 1), which recognizes acetylated α-synuclein whose abundance in biological samples increases in patients with atypical Parkinson's syndrome, and anti-Ac-α-syn antibody B (e.g., an antibody that specifically recognizes methionine 1 of SEQ ID NO: 1), which recognizes acetylated α-synuclein whose abundance in biological samples increases in patients with Parkinson's disease or atypical Parkinson's syndrome. In this case, if the amounts of acetylated α-synuclein detected by antibodies A and B are both elevated above a predetermined reference value, information indicating that the subject is suffering from atypical Parkinson's syndrome is obtained. Alternatively, if only the amount of acetylated α-synuclein detected by antibody B is elevated above a predetermined reference value, information indicating that the subject is suffering from Parkinson's disease is obtained. Furthermore, when the amounts of acetylated α-synuclein detected by antibodies A and B are both below a predetermined reference value, information is acquired that the subject is normal.

[0056] [New biomarkers] Another aspect of the present invention is a diagnostic biomarker for determining whether a patient has Parkinson's disease or atypical Parkinson's syndrome, comprising acetylated α-synuclein. Another aspect of the present invention is a diagnostic biomarker for determining whether a patient has Parkinson's disease or atypical Parkinson's syndrome, comprising a fragment of acetylated α-synuclein having an acetylated amino acid residue. The number of amino acids constituting the acetylated α-synuclein fragment is not limited and may be, for example, 2 to 120, preferably 3 to 100, more preferably 4 to 80, and even more preferably 5 to 60.

[0057] A diagnostic biomarker according to one embodiment of the present invention can be used to diagnose or assist in the diagnosis of whether a subject is suffering from Parkinson's disease or an atypical Parkinson's syndrome. Furthermore, by using two or more diagnostic biomarkers with different acetylated amino acid residues, the biomarkers can be used to diagnose or assist in the diagnosis of whether a subject is normal, suffering from Parkinson's disease, or an atypical Parkinson's syndrome. Furthermore, by using two or more diagnostic biomarkers with different acetylated amino acid residues, the biomarkers can be potentially used to diagnose or assist in the diagnosis of whether a subject is normal, suffering from Parkinson's disease, suffering from multiple system atrophy, or suffering from an atypical Parkinson's syndrome other than multiple system atrophy.

[0058] For example, the presence of atypical Parkinson's syndrome increases the concentration in a biological sample of acetylated α-synuclein in which at least one lysine selected from lysines 12, 34, and 45 of SEQ ID NO: 1 is acetylated. For example, the presence of Parkinson's disease or atypical Parkinson's syndrome increases the concentration in a biological sample of acetylated α-synuclein in which methionine 1 of SEQ ID NO: 1 is acetylated. Therefore, the use of acetylated α-synuclein in which at least one lysine selected from lysines 12, 34, and 45 of SEQ ID NO: 1 and acetylated α-synuclein in which methionine 1 of SEQ ID NO: 1 is acetylated as diagnostic biomarkers enables or aids in the diagnosis of whether a subject is normal, suffers from Parkinson's disease, or suffers from atypical Parkinson's syndrome.

[0059] [Device for measuring acetylated α-synuclein] The present invention also relates to an apparatus (hereinafter sometimes abbreviated as "measuring apparatus") for measuring acetylated α-synuclein in a biological sample collected from a subject. A measuring apparatus according to one embodiment of the present invention will be described in detail with reference to FIG. 1. FIG. 1 is a diagram showing the schematic configuration of a measuring apparatus 20 according to one embodiment of the present invention. The measuring apparatus 20 is an apparatus that acquires a signal resulting from binding between acetylated α-synuclein in a biological sample collected from a subject and an antibody according to one aspect of the present invention.

[0060] 1, measuring device 20 is connected to input unit 30, output unit 31, and storage medium 32. Measuring device 20 includes processing unit (CPU) 21, main storage unit 22, ROM (read only memory) 23, auxiliary storage unit 24, communication interface (I / F) 28, input interface (I / F) 25, output interface (I / F) 26, and media interface (I / F) 27, which are connected to each other via bus 29 so as to be able to communicate data with each other.

[0061] The CPU 21 is a processing unit of the measurement device 20. The CPU 21 executes a computer program stored in the auxiliary storage unit 24 or the ROM 23 and processes acquired data, thereby causing the measurement device 20 to function.

[0062] The ROM 23 is configured by a mask ROM, PROM, EPROM, EEPROM (registered trademark), etc., and stores computer programs executed by the CPU 21 and data used therefor. The CPU 21 may be an MPU 21. The ROM 23 stores a boot program executed by the CPU 21 when the measuring device 20 is started up, as well as programs and settings related to the operation of the hardware of the measuring device 20.

[0063] The main memory unit 22 is configured by RAM (Random Access Memory) such as SRAM or DRAM. The main memory unit 22 is used to read out computer programs recorded in the ROM 23 and the auxiliary memory unit 24. The main memory unit 22 is also used as a working area when the CPU 21 executes these computer programs.

[0064] The auxiliary storage unit 24 is composed of a hard disk, a semiconductor memory device such as a flash memory, an optical disk, etc. The auxiliary storage unit 24 stores various computer programs to be executed by the CPU 21, such as an operating system and application programs, as well as various setting data used to execute the computer programs. Specifically, the auxiliary storage unit 24 stores the measurement program described below, reference values ​​for signal intensity, reference values ​​for measured values ​​of acetylated α-synuclein, etc.

[0065] The communication I / F 28 is composed of a serial interface such as USB, IEEE1394, RS-232C, etc., a parallel interface such as SCSI, IDE, IEEE1284, etc., an analog interface consisting of a D / A converter, an A / D converter, etc., a network interface controller (NIC), etc. Under the control of the CPU 21, the communication I / F 28 receives data from the measurement unit or other external devices, and transmits or displays information stored or generated by the measurement device 20 to the measurement unit or externally as necessary. The communication I / F 28 may communicate with the measurement unit or other external devices via a network.

[0066] The input I / F 25 is configured from, for example, a serial interface such as USB, IEEE1394, RS-232C, etc., a parallel interface such as SCSI, IDE, IEEE1284, etc., and an analog interface including a D / A converter, an A / D converter, etc. In the input I / F 25, the input unit 30 accepts character input, clicks, voice input, etc. The accepted input contents are stored in the main memory unit 22 or the auxiliary memory unit 24.

[0067] The input unit 30 is composed of a touch panel, a keyboard, a mouse, a pen tablet, a microphone, etc., and is used to input text or voice to the measuring device 20. The input unit 30 may be connected to the measuring device 20 from outside, or may be integrated with the measuring device 20.

[0068] The output I / F 26 is configured, for example, by an interface similar to the input I / F 25. The output I / F 26 outputs information generated by the CPU 21 to the output unit 31. The output I / F 26 outputs information generated by the CPU 21 and stored in the auxiliary storage unit 24 to the output unit 31.

[0069] The output unit 31 is composed of, for example, a display, a printer, etc., and displays the measurement results sent from the measurement unit, various operation windows in the measurement device 20, measurement results, etc.

[0070] The media I / F 27 reads, for example, application software stored in the storage medium 32. The read application software is stored in the main memory unit 22 or the auxiliary memory unit 24. The media I / F 27 also writes information generated by the CPU 21 to the storage medium 32. The media I / F 27 writes information generated by the CPU 21 and stored in the auxiliary memory unit 24 to the storage medium 32.

[0071] The storage medium 32 is configured with a flexible disk, a CD-ROM, a DVD-ROM, etc. The storage medium 32 is connected to the media I / F 27 by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, etc. The storage medium 32 may store application programs and the like for the computer to execute operations.

[0072] The CPU 21 may obtain the application software and various settings required for controlling the measuring device 20 via a network instead of reading them from the ROM 23 or the auxiliary storage unit 24. The application program may be stored in the auxiliary storage unit 24 of a server computer on the network, and the measuring device 20 may access this server computer to download the computer program and store it in the ROM 23 or the auxiliary storage unit 24.

[0073] Furthermore, the ROM 23 or the auxiliary storage unit 24 may be installed with an operating system that provides a graphical user interface environment, such as Windows (registered trademark) manufactured and sold by Microsoft Corporation of the United States.

[0074] Processing unit 21 accepts input from the examiner via input unit 30 to acquire signals derived from binding between acetylated α-synuclein in a biological sample and an antibody according to an embodiment of the present invention. Next, signals derived from binding between acetylated α-synuclein and an antibody according to an embodiment of the present invention detected by a measurement unit or the like (described below) are acquired. Processing unit 21 then stores the intensities of these acquired signals in auxiliary storage unit 24 or the like.

[0075] The measurement device 20 may be a personal computer, etc. Furthermore, the measurement device 20 may be built into a measurement unit, which will be described later.

[0076] [System for measuring acetylated α-synuclein in biological samples collected from subjects] The present invention also relates to a system for measuring acetylated α-synuclein in a biological sample collected from a subject (hereinafter, sometimes abbreviated as "measurement system"). The measurement system includes an estimation device 40 and a measurement unit. The measurement system is a system for measuring acetylated α-synuclein in a biological sample collected from a subject.

[0077] The measurement unit includes a detection unit that detects a signal resulting from binding between acetylated α-synuclein in a biological sample and an antibody according to one embodiment of the present invention, and may further include a sample preparation unit for preparing the biological sample, as the case may be. The measurement unit may also be integrated with the estimation device 40. Examples of devices that include an imaging device in the detection unit and are integrated with the estimation device 40 include the Simoa™ HD-1 Analyzer and the Simoa™ HD-X Analyzer.

[0078] [Device for acquiring information to assist in determining whether a person has Parkinson's disease or atypical Parkinson's syndrome] An aspect of the present invention also includes a device (hereinafter sometimes abbreviated as "information acquisition device") that acquires information that assists in determining whether a subject is suffering from Parkinson's disease or atypical Parkinson's syndrome. An information acquisition device according to one embodiment of the present invention is shown in FIG. 2. Information acquisition device 50 is a device that acquires information as to whether a subject is suffering from Parkinson's disease or atypical Parkinson's syndrome based on the detection result of acetylated α-synuclein in a biological sample collected from the subject.

[0079] 2, the information acquisition device 50 is connected to an input unit 60, an output unit 61, and a storage medium 62. The processing unit (CPU) 51, main memory unit 52, ROM (read only memory) 53, auxiliary memory unit 54, communication interface (I / F) 58, input interface (I / F) 55, output interface (I / F) 56, and media interface (I / F) 57 of the information acquisition device 50 are connected to each other via a bus 59 so as to be able to communicate data with each other.

[0080] The details of each component in FIG. 2 are the same as the details of each component in FIG.

[0081] The processing unit 51 receives an input from the input unit 60 by the examiner to acquire a signal resulting from binding between acetylated α-synuclein in a biological sample and an antibody according to an embodiment of the present invention. Next, a signal resulting from binding between acetylated α-synuclein and an antibody according to an embodiment of the present invention, detected by a measurement unit or the like described below, is acquired. The acquired signal intensity is then compared with a reference value of signal intensity stored in the auxiliary memory unit 54 or the like. Based on this comparison, the processing unit 51 determines whether the subject is suffering from Parkinson's disease or atypical parkinsonism. The processing unit 51 stores these determination results in the auxiliary memory unit 54 or the like as information indicating whether the subject is suffering from Parkinson's disease or atypical parkinsonism. The processing unit 51 may output these results to the output unit 61 or store them in a storage medium 62.

[0082] [Computer program for measuring acetylated alpha-synuclein in a subject's biological sample] The present invention also relates to a computer program (hereinafter sometimes abbreviated as "measurement program") for measuring acetylated α-synuclein in a biological sample from a subject. The measurement program is used as a measurement program that controls the operation of a measurement device 20. The measurement program causes a computer to execute the steps of acquiring a signal resulting from binding between acetylated α-synuclein in the biological sample and an antibody according to one embodiment of the present invention, and measuring acetylated α-synuclein in the biological sample from the subject based on the intensity of the signal.

[0083] [Computer program for acquiring information to assist in determining whether a person has Parkinson's disease or atypical Parkinson's syndrome] Another aspect of the present invention also includes a computer program (hereinafter sometimes abbreviated as "information acquisition program") for acquiring information that assists in determining whether a subject is suffering from Parkinson's disease or atypical Parkinsonism. The information acquisition program is used as a measurement program that controls the operation of the information acquisition device 50. The information acquisition program causes a computer to execute a step of acquiring a signal resulting from binding between acetylated α-synuclein in a biological sample and an antibody according to one aspect of the present invention. The information acquisition program then causes the computer to execute a step of acquiring information on whether the subject from whom the biological sample was collected is suffering from Parkinson's disease or atypical Parkinsonism, based on the measured acetylated α-synuclein in the subject's biological sample.

[0084] The computer program may be non-transitory and may be recorded on one or more computer-readable recording media. A computer-readable recording medium having the computer program recorded thereon is also included in one aspect of the present invention. The recording medium may or may not be included in the measurement device or information processing device. In the latter case, the program may be supplied to the measurement device or information processing device via any wired or wireless transmission medium.

[0085] [Method for diagnosing Parkinson's disease or atypical Parkinson's syndrome] Another aspect of the present invention is a method for diagnosing Parkinson's disease or atypical Parkinsonism. The method includes a step of diagnosing whether a patient is suffering from Parkinson's disease or atypical Parkinsonism based on acetylated α-synuclein detected in a biological sample collected from the patient by the measurement method according to one aspect of the present invention. The method includes diagnosis by a physician or other qualified medical professional.

[0086] In the diagnostic step, the detected acetylated α-synuclein is compared with a predetermined reference value. If the detected acetylated α-synuclein is significantly increased or decreased compared to the predetermined reference value, the subject is diagnosed as suffering from Parkinson's disease or atypical parkinsonism. Examples of the predetermined reference value are the same as those described in the "Information Acquisition Method."

[0087] By diagnosing whether a subject is suffering from Parkinson's disease or atypical Parkinson's syndrome, the progression of the disease, the progress of treatment, or the effect of treatment can be objectively observed and evaluated.

[0088] Methods for assisting patient diagnosis

[0013] Another aspect of the present invention is a method for assisting in the diagnosis of a patient. One aspect of the method for assisting in the diagnosis of a patient includes a step of obtaining data suggesting whether the biological sample is derived from a patient suffering from Parkinson's disease or a patient suffering from atypical Parkinsonism, based on acetylated alpha-synuclein in the biological sample collected from the patient detected by the measurement method according to one aspect of the present invention. Another aspect of the method for assisting in the diagnosis of a patient includes a step of obtaining data suggesting whether the biological sample is derived from a normal subject, a patient suffering from Parkinson's disease, or a patient suffering from atypical Parkinsonism, based on acetylated alpha-synuclein in the biological sample collected from the patient detected by the measurement method according to one aspect of the present invention.

[0089] 〔summary〕 The antibody according to embodiment 1 of the present invention is an antibody that recognizes an acetylated epitope containing an acetylated amino acid residue in the amino acid sequence shown in SEQ ID NO:1.

[0090] The antibody according to aspect 2 of the present invention may be the antibody according to aspect 1, wherein the acetylated epitope comprises an acetylated amino acid residue in the amino acid sequence at positions 1 to 60 of the amino acid sequence shown in SEQ ID NO:1.

[0091] The antibody according to aspect 3 of the present invention, in aspect 1 or 2 above, preferably does not recognize an epitope that does not contain acetylated amino acid residues in the amino acid sequence shown in SEQ ID NO:1.

[0092] A measurement method according to aspect 4 of the present invention is a method for measuring acetylated alpha-synuclein, comprising a reaction step of binding the antibody according to any one of aspects 1 to 3 to acetylated alpha-synuclein in a biological sample collected from a subject, and a detection step of detecting the acetylated alpha-synuclein bound to the antibody.

[0093] A fifth aspect of the present invention relates to a measurement method in the fourth aspect, wherein the biological sample is a blood sample, saliva, cerebrospinal fluid, urine, or a tissue extract.

[0094] A sixth aspect of the present invention relates to a measurement method in the fifth aspect, wherein the blood sample is serum or plasma.

[0095] In the measurement method according to aspect 7 of the present invention, in aspect 4 or 5, the acetylated α-synuclein may be detected by immunoassay or immunoprecipitation-mass spectrometry in the detection step.

[0096] A kit according to an eighth aspect of the present invention is a kit for measuring acetylated α-synuclein, comprising one or more antibodies according to any one of the first to third aspects.

[0097] The kit according to aspect 9 of the present invention may be the kit of aspect 8, further comprising an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 and recognizes an epitope different from the acetylated epitope.

[0098] An information acquisition method according to aspect 10 of the present invention is an information acquisition method for acquiring information on whether a subject is suffering from Parkinson's disease or atypical Parkinsonism, comprising a step of measuring the concentration of acetylated α-synuclein in a biological sample collected from the subject.

[0099] The information acquisition method according to an eleventh aspect of the present invention may be the same as that of the tenth aspect, in which information is acquired as to whether the subject is normal, suffers from Parkinson's disease, or suffers from atypical Parkinsonism.

[0100] The information acquisition method according to aspect 12 of the present invention is an information acquisition method that acquires information on whether a subject is suffering from Parkinson's disease, atypical Parkinson's syndrome, or neither Parkinson's disease nor atypical Parkinson's syndrome, based on the amount of acetylated α-synuclein detected by the measurement method of any one of aspects 4 to 7.

[0101] The information acquisition method according to aspect 13 of the present invention may be the same as that of aspect 12, and may acquire information as to whether the subject is normal, suffers from Parkinson's disease, suffers from atypical Parkinson's syndrome, or suffers from neither Parkinson's disease nor atypical Parkinson's syndrome.

[0102] A diagnostic biomarker according to aspect 14 of the present invention is a diagnostic biomarker for determining whether a subject is suffering from Parkinson's disease or atypical Parkinsonism, which comprises acetylated α-synuclein.

[0103] A measuring device according to aspect 15 of the present invention is a device for measuring acetylated alpha-synuclein in a biological sample collected from a subject, the device comprising a processing unit, which acquires a signal resulting from the binding between acetylated alpha-synuclein in the biological sample collected from the subject and the antibody according to any one of aspects 1 to 3.

[0104] A computer program according to aspect 16 of the present invention is a computer program for measuring acetylated alpha-synuclein in a biological sample from a subject, which causes a computer to execute the step of acquiring a signal resulting from binding between acetylated alpha-synuclein in the biological sample and the antibody of any one of aspects 1 to 3, wherein the biological sample is collected from the subject.

[0105] A recording medium according to a seventeenth aspect of the present invention is a computer-readable recording medium having the program according to the sixteenth aspect recorded thereon.

[0106] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents described in this specification are incorporated by reference. [Example]

[0107] [Materials for Evaluation Examples 1 and 2] Capture beads were prepared using beads from Quanterix's Simoa™ Homebrew Assay Development Kit according to the attached protocol. Quanterix's Dye-Encoded Helper Beads were used as non-capture beads. The mouse monoclonal antibody Syn-1 (Transduction Laboratories) was used as the capture antibody. The antigen peptide of the Syn-1 antibody corresponds to amino acids 91 to 99 of SEQ ID NO: 1. Three anti-Ac-α-syn rabbit monoclonal antibodies (SA17E40, SB20F51, and SB20F56) produced by Cell Engineering Institute, Inc. were used as detection antibodies. All three anti-Ac-α-syn rabbit monoclonal antibodies specifically recognize acetylation of lysines 12, 34, and 45 of the amino acid sequence of SEQ ID NO: 1. To generate three anti-Ac-α-syn rabbit monoclonal antibodies, rabbits were immunized with the synthetic peptide α-Syn K34Ac(29-38), corresponding to amino acids 29-38 of SEQ ID NO: 1, in which lysine 34 was acetylated. Lymphocytes were collected from the rabbits and positive cells reacting with the target antigen were selected. Antibody genes were isolated from the selected lymphocytes and transfected into HEK293 cells. Antigen-specific positive clones were selected by ELISA (first ELISA) using the antibody-secreting culture supernatant. Plasmids containing the Ac-α-syn antibody genes of the selected positive clones (SA17E40, SB20F51, and SB20F56) were constructed. The constructed plasmids were then transfected into HEK293 cells, and the reproducibility of the first ELISA was confirmed by ELISA using the antibody-secreting culture supernatant.

[0108] Hereinafter, the method for measuring Ac-α-syn using the above-mentioned Syn-1 antibody as a capture antibody and any of the above-mentioned SA17E40, SB20F51 or SB20F56 as a detection antibody may be abbreviated as the measurement method of Example 1.

[0109] The biotinylated detection antibody was prepared according to the protocol of the Simoa™ Homebrew Assay Development kit.

[0110] [Measurement of Ac-α-syn by sandwich ELISA] In this example, acetylated α-synuclein (Ac-α-syn) was measured by sandwich ELISA. The Simoa™ machine from Quanterix was used as the sandwich ELISA device, and measurements were performed according to the manufacturer's protocol. The steps for measuring Ac-α-syn using the Simoa™ machine are described below.

[0111] First, the capture antibody bound to the capture beads was allowed to bind to Ac-α-syn in the blood. Then, a biotinylated detection antibody was further bound to the capture antibody-bound Ac-α-syn, forming an immune complex between the capture antibody, Ac-α-syn, and the biotinylated detection antibody on the capture beads. The capture beads were then reacted with streptavidin conjugated with β-galactosidase. After the reaction, the capture beads were reacted with a β-galactosidase substrate (resorufin β-D-galactopyranoside (RGP)) in the presence of non-capture beads. After the RGP reaction, the capture beads were applied to a Simoa™ Disk. The applied beads were then placed one by one in each well of an array containing multiple femtoliter-sized wells. Each bead in the well was imaged using the Simoa™ HD-1 Analyzer, and Ac-α-syn was quantified by measuring the fluorescence intensity (AEB) and counting the number of beads with high fluorescence intensity.

[0112] [Evaluation Example 1] The prepared anti-Ac-α-syn antibodies (SA17E40, SB20F51, and SB20F56) were evaluated. Anti-Ac-α-syn antibodies (SA17E40, SB20F51, and SB20F56) in the culture supernatant of HEK293 cells transfected with the above plasmids were added to an ELISA plate on which the following six antigen peptides (1 μg / mL, 50 μL / well) had been immobilized, and color development was performed using anti-rabbit IgG and TMBZ color reagent. The reaction curves for each antibody are shown in Tables 1 to 3 and Figures 3 to 5. The values ​​in Tables 1 to 3 indicate absorbance (450 nm).

[0113] Antigen 1: BSA-MBS-α-Syn K34Ac(29-38): α-Syn 29-38 partial peptide with acetylation at K34 Antigen 2: BSA-MBS-α-Syn(29-38): a partial peptide of α-Syn29-38 with no acetylation at K34 Antigen 3: BSA-MBS-α-SynK60Ac(55-64): a partial peptide of α-Syn 55-64 in which K60 is acetylated Antigen 4: BSA-MBS-α-SynK12Ac(7-16): α-Syn7-16 partial peptide with K12 acetylation Antigen 5: BSA-MBS-α-SynK45Ac(40-49): a partial peptide of α-Syn40-49 in which K45 is acetylated Antigen 6: BSA: Carrier protein alone (non-α-Syn protein)

[0114] The numbers in parentheses for each antigen above indicate the amino acid number of α-syn (SEQ ID NO: 1), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) is a crosslinker, and BSA (bovine serum albumin) is a carrier protein that enhances immunoreactivity. K12, K34, and K45 represent the 12th, 34th, and 45th lysine residues of α-syn (SEQ ID NO: 1), respectively.

[0115] [Table 1]

[0116] [Table 2]

[0117] [Table 3]

[0118] As shown in Tables 1 to 3 and Figures 3 to 5, each of the anti-Ac-α-syn antibodies prepared recognized multiple acetylated α-syn fragment peptides (antigens 1, 3, 4, or 5) including acetylation at K34 (antigen 1), but did not react with non-acetylated α-syn fragments (antigen 2) or BSA (antigen 6).

[0119] [Evaluation Example 2] Plasma samples were collected from four PD patients and four MSA patients who had been clinically diagnosed at the Department of Neurology, Kyoto Prefectural University of Medicine, and whose subsequent clinical course was consistent with PD or MSA. The amount of Ac-α-syn in the patient-derived biological samples was then measured using the measurement method of Example 1. The plasma was diluted four-fold with Sample Diluent before measurement. Buffer was used as a blank.

[0120] The results of measuring Ac-α-syn concentrations in patient-derived biological samples are shown in Figures 6 to 8. As shown in Figures 6 to 8, whether SA17E40, SB20F51, or SB20F56 was used as the detection antibody, a clear difference was observed in the Ac-α-syn measurements in the biological samples between MSA and PD patients, with higher Ac-α-syn concentrations in MSA patients than in PD patients. In particular, when SA17E40 was used as the detection antibody, MSA and PD patients could be clearly distinguished based on the Ac-α-syn measurements in the patient-derived biological samples (Figure 6).

[0121] [Materials for Evaluation Examples 3 and 4] Capture beads were prepared using beads from Quanterix's Simoa™ Homebrew Assay Development kit according to the attached protocol. Quanterix's Dye-Encoded Helper Beads were used as non-capture beads. The mouse monoclonal antibody Syn-1 (Transduction Laboratories) was used as the capture antibody. The antigen peptide of the Syn-1 antibody corresponds to amino acids 91 to 99 of the amino acid sequence of SEQ ID NO: 1. Two anti-Ac-α-syn rabbit monoclonal antibodies (S08H8 and S08H17) produced by Cell Engineering Institute, Inc. were used as detection antibodies. Both anti-Ac-α-syn rabbit monoclonal antibodies specifically recognize acetylation of the first methionine in the amino acid sequence of SEQ ID NO: 1. To generate two anti-Ac-α-syn rabbit monoclonal antibodies, rabbits were immunized with the synthetic peptide α-Syn M1Ac(1-6)+3K, which corresponds to amino acid residues 1-6 of SEQ ID NO: 1, with the first methionine acetylated, followed by the addition of three lysine residues to the C-terminus. Lymphocytes were collected from the rabbits and positive cells reacting with the target antigen were selected. Antibody genes were isolated from the selected lymphocytes and transfected into HEK293 cells. Antigen-specific positive clones were selected by ELISA (1st ELISA) using the antibody-secreting culture supernatant. Plasmids carrying the Ac-α-syn antibody gene from the selected positive clones (S08H8 and S08H17) were constructed. The constructed plasmids were then transfected into HEK293 cells, and the reproducibility of the 1st ELISA was confirmed by ELISA using the antibody-secreting culture supernatant.

[0122] Hereinafter, the method for measuring Ac-α-syn using the above-mentioned Syn-1 antibody as a capture antibody and either S08H8 or S08H17 as a detection antibody may be abbreviated as the measurement method of Example 2.

[0123] [Evaluation Example 3] The anti-Ac-α-syn antibodies (S08H8, S08H17) prepared were evaluated. Anti-Ac-α-syn antibodies (S08H8, S08H17) in the culture supernatant of HEK293 cells transfected with the above-mentioned plasmids were added to an ELISA plate on which the following four antigen peptides (1 μg / mL, 50 μL / well) had been immobilized, and color development was carried out using anti-rabbit IgG and TMBZ color development reagent. The reaction curves for each antibody are shown in Tables 4-5 and Figures 9-10. The values ​​in Tables 1-3 indicate absorbance (450 nm).

[0124] Antigen 1: BSA-CL-α-Syn M1Ac(1-6)+3K: α-Syn 1-6 partial peptide with acetylated M1 and three lysine residues added to the C-terminus Antigen 2: BSA-CL-α-Syn(1-6)+3K: α-Syn1-6 partial peptide with no M1 acetylation and three lysine residues added to the C-terminus Antigen 3: BSA-CL-α-Syn M1Ac(1-6): α-Syn 1-6 partial peptide with acetylated M1 Antigen 4: BSA-CL-α-Syn(1-6): a partial peptide of α-Syn(1-6) with no acetylation of M1

[0125] The numbers in parentheses for each antigen above indicate the amino acid number of α-syn (SEQ ID NO: 1), CL (crosslinker) is a crosslinking agent, BSA is a carrier protein that enhances immunoreactivity, and M1 indicates the first methionine residue of α-syn (SEQ ID NO: 1).

[0126] [Table 4]

[0127] [Table 5]

[0128] As shown in Tables 4-5 and Figures 9-10, each of the anti-Ac-α-syn antibodies prepared recognized multiple acetylated α-syn fragment peptides (either antigen 1 or 3) including acetylated M1 (antigen 1), but did not react with non-acetylated α-syn fragments (antigens 2 and 4).

[0129] [Evaluation Example 4] Plasma samples were collected from four PD and four MSA patients who had been clinically diagnosed at the Department of Neurology, Kyoto Prefectural University of Medicine, and whose subsequent clinical course was consistent with PD or MSA. Plasma samples were also collected from one age-matched healthy elderly (HC). The amount of Ac-α-syn in the patient and HC biological samples was measured using the measurement method of Example 2. Specifically, Ac-α-syn was measured by sandwich ELISA using the same procedure as in Evaluation Example 2, except that the combination of detection and capture antibodies was changed. Plasma was diluted 4-fold with Sample Diluent before measurement. Buffer was used as a blank.

[0130] The results of measuring Ac-α-syn concentrations in patient-derived biological samples are shown in Figures 11 and 12. As shown in Figures 11 and 12, whether S08H8 or S08H17 was used as the detection antibody, clear differences were observed in the Ac-α-syn measurements in the biological samples between HC patients and MSA and PD patients, with both MSA and PD patients having higher Ac-α-syn concentrations than HC patients. In other words, the Ac-α-syn measurements in the biological samples from MSA and PD patients clearly distinguished MSA and PD patients from HC patients.

[0131] As described above, the measurement method of the present example makes it possible to quantify Ac-α-syn in blood, such as plasma, which was previously impossible. Furthermore, this evaluation example demonstrated that Ac-α-syn can be a biomarker capable of distinguishing between Parkinson's disease and atypical parkinsonian syndromes, such as MSA. Until now, no blood biomarker capable of distinguishing between PD and MSA existed internationally. Differential diagnosis using blood tests with Ac-α-syn as a blood biomarker is not only noninvasive and efficient (high-throughput), but can also be performed easily and inexpensively. Therefore, differential diagnosis using blood tests with Ac-α-syn as a blood biomarker can be performed at all medical institutions, from primary care physicians to flagship hospitals. [Industrial Applicability]

[0132] The antibody of the present invention can provide information that can be used for biochemical diagnosis of whether a patient is suffering from Parkinson's disease or atypical Parkinsonism, and can be used for pharmaceutical purposes, etc. [Explanation of symbols]

[0133] 20 Measuring Equipment 21, 51 Processing unit (CPU) 22, 52 Main memory 23, 53 ROM 24, 54 Auxiliary storage 25, 55 input I / F 26, 56 output I / F 27, 57 Media I / F 28, 58 Communication I / F Buses 29 and 59 30, 60 Input section 31, 61 Output section 32, 62 Storage medium 50 Information acquisition device

Claims

1. An antibody that recognizes an acetylated epitope containing an acetylated amino acid residue in the amino acid sequence shown in SEQ ID NO:

1.

2. The antibody according to claim 1, wherein the acetylated epitope is an epitope comprising acetylated amino acid residues in the amino acid sequence of positions 1 to 60 of the amino acid sequence shown in SEQ ID NO:

1.

3. The antibody of claim 1, which does not recognize an epitope that does not contain acetylated amino acid residues in the amino acid sequence shown in SEQ ID NO:

1.

4. A reaction step of binding the antibody according to claim 1 to acetylated α-synuclein in a biological sample collected from a subject; and A method for measuring acetylated α-synuclein, comprising a detection step of detecting the acetylated α-synuclein bound to the antibody.

5. The measurement method according to claim 4, wherein the biological sample is a blood sample, saliva, cerebrospinal fluid, urine, or tissue extract.

6. The measurement method according to claim 5 , wherein the blood sample is serum or plasma.

7. The method according to claim 4, wherein the acetylated α-synuclein is detected by immunoassay or immunoprecipitation-mass spectrometry in the detection step.

8. A kit for measuring acetylated α-synuclein, comprising one or more antibodies according to any one of claims 1 to 3.

9. The kit according to claim 8, further comprising an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 and recognizes an epitope different from the acetylated epitope.

10. An information acquisition method for acquiring information on whether a subject is suffering from Parkinson's disease or atypical Parkinson's syndrome, comprising a step of measuring the concentration of acetylated α-synuclein in a biological sample collected from the subject.

11. The information acquisition method according to claim 10, wherein information is acquired as to whether a subject is normal, suffers from Parkinson's disease, or suffers from atypical Parkinson's syndrome.

12. An information obtaining method for obtaining information on whether a subject is suffering from Parkinson's disease, atypical Parkinson's syndrome, or neither Parkinson's disease nor atypical Parkinson's syndrome, based on the amount of acetylated α-synuclein detected by the measurement method according to any one of claims 4 to 7.

13. The information acquisition method according to claim 12, wherein information is acquired as to whether a subject is normal, suffers from Parkinson's disease, suffers from atypical Parkinson's syndrome, or suffers from neither Parkinson's disease nor atypical Parkinson's syndrome.

14. A diagnostic biomarker for determining whether a patient has Parkinson's disease or atypical Parkinsonism, comprising acetylated α-synuclein.

15. An apparatus for measuring acetylated α-synuclein in a biological sample collected from a subject, comprising: The apparatus comprises a processing unit; The processing unit acquires a signal derived from the binding between acetylated α-synuclein in a biological sample collected from a subject and the antibody according to any one of claims 1 to 3.

16. On the computer, A step of acquiring a signal resulting from binding between acetylated α-synuclein in a biological sample and the antibody according to any one of claims 1 to 3, A computer program for measuring acetylated α-synuclein in a biological sample from a subject, the computer program causing the computer to perform the steps of: the biological sample being collected from the subject.

17. A computer-readable recording medium on which the program according to claim 16 is recorded.

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