Method and device for estimating accumulation amount of TDP-43 in brain of subject

Abstract

The present disclosure pertains to a method for estimating the accumulation amount of TDP-43 in the brain. An epitope of one antibody among a capture antibody and a detection antibody used in said method includes a peptide up to the 203rd to 209th amino acids of the amino acid sequence represented by SEQ ID NO: 1. An epitope of the other antibody is: an epitope included in a peptide composed of the 261st to 414th amino acids of the amino acid sequence represented by SEQ ID NO: 1; or an epitope including a peptide which is composed of the 404th to 410th amino acids and in which the 409th and 410th serines are phosphorylated.

Description

Method and apparatus for estimating the amount of TDP-43 accumulated in the brain of a subject

[0001] The present invention relates to a method and apparatus for detecting TDP-43 in a biological sample collected from a subject and estimating the amount of TDP-43 accumulated in the brain of the subject.

[0002] TDP-43 is known as an RNA-binding protein and is considered to be one of the candidate biomarkers for diseases such as frontotemporal lobe dementia (FTLD) and amyotrophic lateral sclerosis (ALS).

[0003] For example, Patent Document 1 and Non-Patent Document 1 disclose a method for measuring TDP-43 using Simoa (trademark), which is known as an ultra-sensitive digital ELISA device.

[0004] Non-Patent Document 2 reports that TDP-43 accumulates in the central and peripheral nervous systems in cranial nerve diseases such as ALS, frontotemporal lobar degeneration due to TDP-43 accumulation (FTLD-TDP), and limbic-predominant age-related TDP-43 encephalopathy (LATE). There is a need for the development of a method for estimating the amount of TDP-43 accumulated in the brain for the diagnosis of these cranial nerve diseases.

[0005] Non-Patent Document 3 discloses that the ratio of glial fibrillary acidic protein / neurofilament light chain (GFAP / NfL ratio) in the blood can be used to differentiate between frontotemporal lobar degeneration due to tau accumulation (FTLD-tau) and FTLD-TDP.

[0006] Japanese Patent No. 6961278

[0007] Kasai T, et al., Ann Clin Transl Neurol. 2019; 6(12):2489-2502Kapasi A, et al., Neurology. 2020; 95(14): e1951-e1962Cousins ​​KAQ, et al., JAMA Neurol. 2022; 79(11): 1155-1164

[0008] It is known that GFAP and NfL are elevated in the presence of Alzheimer's disease (AD) pathology. Therefore, in elderly people who frequently have AD pathology, it is not possible to distinguish between FTLD-tau and FTLD-TDP using the technique disclosed in Non-Patent Document 3. For example, even in 70-year-olds with normal cognitive function, amyloid accumulation is observed in 25%.

[0009] Therefore, there is no method that can estimate the amount of TDP-43 accumulated in the brain, and the development of such a method is desired.

[0010] An object of one aspect of the present invention is to provide a method, an apparatus, etc. that can estimate the amount of TDP-43 accumulated in the brain.

[0011] In order to solve the above problems, the present invention includes the following aspects. a preparation step of forming an immune complex between TDP-43 in a biological sample collected from the subject, a capture antibody, and a detection antibody on capture beads in the presence of non-capture beads, thereby preparing a measurement sample containing the non-capture beads and capture beads to which the immune complex is bound; a detection step of detecting a signal derived from the immune complex in the measurement sample; and an estimation step of estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, wherein the non-capture beads do not bind to the immune complex, one of the detection antibody and the capture antibody has an epitope comprising a peptide consisting of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the other of the detection antibody and the capture antibody has an epitope comprising an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated. a processing unit, which acquires a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads bound to the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample collected from a subject, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, and estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, wherein the non-capture beads do not bind to the immune complex, one of the epitope of the detection antibody and the capture antibody comprises a peptide consisting of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated.a device for obtaining information on whether or not a subject is suffering from a TDP-43 proteinopathy based on the intensity of a signal derived from TDP-43 in a biological sample collected from the subject, the device comprising a processing unit, wherein the processing unit: obtains a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads to which the immune complex is bound, the measurement sample being prepared by forming an immune complex between TDP-43 in the biological sample collected from the subject, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads; estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal; and obtains information on whether or not the subject is suffering from a TDP-43 proteinopathy based on the estimated amount of TDP-43 accumulated in the brain of the subject; the non-capture beads do not bind to the immune complex; the epitope of one of the detection antibody and capture antibody comprises the peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1; The epitope of the other of the detection antibody and capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated.An apparatus for obtaining information on whether a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the intensity of a signal derived from TDP-43 in a biological sample collected from the subject, the apparatus comprising a processing unit, the processing unit: obtains a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads to which the immune complex is bound, the measurement sample being prepared by forming an immune complex on capture beads between TDP-43 in the biological sample collected from the subject, a detection antibody, and a capture antibody in the presence of non-capture beads; estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal; and obtains information on whether the subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the estimated amount of TDP-43 accumulated in the brain of the subject; the non-capture beads do not bind to the immune complex, an epitope of one of the detection antibody and the capture antibody comprises a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1; and an epitope of the other of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated.a step of acquiring a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads bound with the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the non-capture beads do not bind to the immune complex, the epitope of one of the detection antibody and the capture antibody comprises a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated; and a step of estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal. A computer program for estimating the amount of TDP-43 accumulated in the brain of a subject, which executes the above steps.a step of acquiring a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads bound with the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the non-capture beads do not bind to the immune complex, the epitope of one of the detection antibody and the capture antibody comprises a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated; and a step of estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal. and obtaining information on whether or not the subject from whom the biological sample was collected is affected with TDP-43 proteinopathy, based on the estimated amount of TDP-43 accumulated in the subject's brain.a step of acquiring a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads bound with the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the non-capture beads do not bind to the immune complex, the epitope of one of the detection antibody and the capture antibody comprises a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated; and a step of estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal. A computer program for obtaining information on whether or not a subject from whom the biological sample was collected is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, the computer program executing the steps of: obtaining information on whether or not a subject from whom the biological sample was collected is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the estimated amount of accumulated TDP-43 in the subject's brain.

[0012] According to one aspect of the present invention, a method, an apparatus, etc. can be realized that can estimate the amount of TDP-43 accumulated in the brain.

[0013] 1 is a diagram showing the results of evaluation example 1. FIG. 2 is a diagram showing the results of evaluation example 2. FIG. 3 is a diagram showing the configuration of a main part of an estimation device according to an aspect of the present invention. FIG. 4 is a diagram showing the configuration of a main part of an information acquisition device according to an aspect of the present invention.

[0014] 1. Method for Estimating the Amount of TDP-43 Accumulated in the Brain of a Subject A method for estimating the amount of TDP-43 accumulated in the brain of a subject (hereinafter, sometimes abbreviated as "estimation method") according to one embodiment of the present invention is a method for estimating the amount of TDP-43 accumulated in the brain of a subject by detecting TDP-43 in a biological sample collected from the subject. By this estimation method, TDP-43 in the brain of the subject can be estimated.

[0015] TDP-43, an abbreviation for TAR DNA-binding protein 43, is an RNA / DNA-binding protein with two RNA recognition motifs. SEQ ID NO: 1 is the amino acid sequence of human TDP-43. TDP-43 is involved in RNA metabolism, including the regulation of transcription, translation, and splicing. In normal neurons, TDP-43 is primarily localized in the nucleus. However, in neurons from patients with TDP-43 proteinopathies such as FTLD and ALS, phosphorylated TDP-43 is localized in the cytoplasm or neurites.

[0016] For example, an example of human-derived TDP-43 is the amino acid sequence shown in SEQ ID NO: 1 (amino acid sequence registered as NCBI Reference Sequence NP_031401).

[0017] The estimation method according to one embodiment of the present invention can estimate at least one of the amount of non-phosphorylated TDP-43 and the amount of phosphorylated TDP-43 in the brain of a subject. Unless otherwise specified, "TDP-43" herein refers to both non-phosphorylated TDP-43 and phosphorylated TDP-43. Phosphorylated TDP-43 is not limited as long as the TDP-43 is phosphorylated. Examples of phosphorylated TDP-43 include, but are not limited to, TDP-43 in which at least one of the serine at position 403 (Ser403), serine at position 404 (Ser404), serine at position 409 (Ser409), and serine at position 410 (Ser410) in the amino acid sequence registered as NCBI Reference Sequence NP_031401 is phosphorylated.

[0018] 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.

[0019] The biological sample collected from the subject is not particularly limited as long as it can contain TDP-43. Examples of biological samples include blood samples, saliva, spinal fluid (cerebrospinal fluid), urine, and tissue extract samples. Examples of tissue extracts include liquids extracted from tissues such as the brain, peripheral nerves, muscles, spinal cord, and dorsal root ganglia.

[0020] The biological sample is preferably a blood sample, in that it is minimally invasive and allows for more accurate estimation of the amount of TDP-43 accumulated in the brain. Blood samples include whole blood, plasma, serum, etc., and the blood sample is preferably plasma or serum, and more preferably plasma. Plasma can be separated from whole blood collected in the form of heparin salt (preferably sodium salt), citrate salt (preferably sodium salt), or ethylenediaminetetraacetic acid (EDTA) salt (preferably sodium salt or potassium salt). It is more preferable that the plasma be plasma separated from whole blood collected in the form of EDTA salt or heparin salt.

[0021] The estimation method according to one aspect of the present invention includes a preparation step, a detection step, and an estimation step. Each step will be described below.

[0022] (Preparation step) In the preparation step, an immune complex is formed on the capture beads between TDP-43 in a biological sample from a subject, the capture antibody, and the detection antibody in the presence of non-capture beads, to prepare a measurement sample containing the non-capture beads and the capture beads to which the immune complex is bound.

[0023] <Antibody> The epitope of one of the detection antibody and capture antibody comprises the peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1. Hereinafter, an antibody having this epitope may be referred to as "TDP-43 antibody 1." Examples of TDP-43 antibody 1 include 60019-2-Ig (Proteintech).

[0024] The epitope of the other of the detection antibody and capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated. Hereinafter, an antibody having an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1 may be referred to as "TDP-43 antibody 2." Furthermore, an antibody having an epitope containing a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated may be referred to as "phosphorylated TDP-43 antibody." Examples of TDP-43 antibody 2 include 12892-1-AP (Proteintech).

[0025] In estimating non-phosphorylated TDP-43 in the brain, it is preferable that one of the capture antibody and the detection antibody is TDP-43 antibody 1, and the other is TDP-43 antibody 2. In measuring phosphorylated TDP-43, it is preferable that one of the capture antibody and the detection antibody is TDP-43 antibody 1, and the other is a phosphorylated TDP-43 antibody.

[0026] The detection antibody and capture antibody may be any of polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab)2, etc.). The class and subclass of immunoglobulin are not particularly limited. Antibodies selected from antibody libraries by phage display or other methods may also be used. The detection antibody and capture antibody may also be commercially available antibodies.

[0027] As used herein, the term "capture antibody" refers to an antibody that binds to TDP-43 and is immobilized on beads, which will be described later. The immobilization on beads may be indirect, using a substance such as avidin (or streptavidin) or biotin. As used herein, the term "detection antibody" refers to an antibody that binds to TDP-43 and preferably has a labeling substance, which will be described later.

[0028] <Beads> The beads are not limited as long as they have properties that allow individual beads to be spatially separated. The beads may be provided in a form that allows them to be spatially separated into multiple locations (e.g., wells). For example, the beads may be shaped like spheres, disks, rings, cubes, etc. The beads may also be microparticles (e.g., multiple particles suspended in a liquid), nanotubes, etc.

[0029] The size or shape of the beads can be designed appropriately. For example, bead shapes include spheres, cubes, ellipsoids, tubes, etc. The average diameter (if substantially spherical) or average maximum cross-sectional dimension (if other shapes) of the beads may be about 0.1 μm or more, about 1 μm or more, about 10 μm or more, about 100 μm or more, or about 1 mm or more. The average diameter or maximum outer dimension of the beads may be about 0.1 μm to about 100 μm, about 1 μm to about 100 μm, about 10 μm to about 100 μm, about 0.1 μm to about 1 mm, about 1 μm to about 10 mm, or about 0.1 μm to about 10 μm. The average diameter or average maximum cross-sectional dimension of the beads used in this embodiment is the arithmetic mean value of the diameter / maximum cross-sectional dimension of the beads.

[0030] The average maximum cross-sectional dimension of the beads can be measured using, for example, laser light scattering, microscopy, sieve analysis, electrical resistance, or other known techniques. The average diameter of the beads is the volume-based median diameter measured using a particle size distribution analyzer based on laser diffraction and scattering. Examples of such particle size distribution analyzers include the "Microtrac MT3000II" manufactured by Nikkiso Co., Ltd.

[0031] The beads are insoluble or substantially insoluble in the solvent or solution used in the assay. The beads may be porous or substantially porous, hollow, partially hollow, etc. Preferably, the beads are non-porous solids or substantially non-porous solids (e.g., essentially pore-free). The beads may substantially absorb or absorb the solvent. Preferably, the beads are non-absorbing or substantially non-absorbing the solvent.

[0032] Furthermore, the material of the beads can be selected from plastics or synthetic polymers (e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose, etc.), naturally occurring polymers (latex rubber, polysaccharides), metals or metal compounds (e.g., metal compounds including gold, silver, steel, aluminum, copper, etc.), inorganic glass, silica, and mixtures thereof. Preferably, the beads comprise a magnetic material. Furthermore, the beads are preferably optically detectable.

[0033] The beads may be commercially available beads, for example, carboxyl-functionalized paramagnetic beads (diameter 2.7 μm) from Agilent Technologies.

[0034] The buffer used for the beads preferably contains a chelating agent, more preferably EDTA, in order to stabilize the antigen-antibody reaction. The addition of EDTA can reduce the rate of measurement errors. A chelating agent is preferably added to the buffer used for the capture beads in order to reduce the rate of measurement errors (particularly the rate of measurement errors caused by bead aggregation that makes measurement impossible).

[0035] Furthermore, in order to improve the sensitivity of the antigen-antibody reaction and reduce the background during detection of the antigen-antibody reaction, it is preferable that a mammal-derived serum component or a blocking agent be added to the buffer solution (sample buffer) used to dilute the biological sample. Examples of mammal-derived serum components or blocking agents include albumin, casein, and skim milk.

[0036] As used herein, capture beads are beads capable of immobilizing a complex containing TDP-43. For example, beads to which a molecule capable of binding to TDP-43 (hereinafter also referred to as a "capture molecule") is directly or indirectly immobilized can be used as capture beads. Examples of capture molecules include the above-mentioned capture antibodies.

[0037] The method for preparing capture beads is not limited as long as the capture molecule can be bound to the beads. For example, capture beads may be prepared using a commercially available kit such as the Simoa™ Homebrew Assay Development kit from Quanterix. The capture beads may be blocked with albumin, casein, skim milk, or the like to suppress nonspecific binding.

[0038] As used herein, non-capture beads are beads that do not substantially bind to TDP-43. Non-capture beads are not provided with a capture molecule. The material of the beads used for the non-capture beads may be the same as or different from that of the beads used for the capture beads. Furthermore, for example, Dye-Encoded Helper Beads from Quanterix may be used as non-capture beads. The non-capture beads may be blocked with albumin, casein, skim milk, or the like to suppress non-specific binding.

[0039] The method for forming an immune complex between TDP-43 in a biological sample, a capture antibody, and a detection antibody on capture beads is not limited as long as this embodiment can be realized. For example, the immune complex can be formed as follows. First, TDP-43 in a biological sample is bound to the capture antibody, and TDP-43 is bound to (preferably captured by) the capture beads. Next, the detection antibody is bound to the TDP-43 captured by the capture antibody to form an immune complex.

[0040] Alternatively, an immune complex may be formed by forming an immune complex between TDP-43 in a biological sample, a capture antibody, and a detection antibody in a buffer solution and then binding the immune complex to capture beads. For example, the immune complex can be immobilized on the capture beads by contacting a complex of a biotin-labeled capture antibody, TDP-43, and a detection antibody with beads on which avidin or streptavidin is immobilized.

[0041] The immune complex is formed in the presence of non-capture beads. When preparing a measurement sample, the ratio of the number of capture beads to the number of non-capture beads is preferably 2.3 or more, more preferably 3 or more, when the number of capture beads is 1. Furthermore, the ratio of the number of capture beads to the number of non-capture beads is preferably 9 or less when the number of capture beads is 1. When the ratio of the number of capture beads to the number of non-capture beads is within the above range, TDP-43 can be detected with higher sensitivity.

[0042] The total number of beads (capture beads and non-capture beads) mixed with the biological sample or diluted biological sample may be, for example, 400,000 or more for 90 μL of measurement sample, or 500,000 or less for 90 μL of measurement sample.

[0043] The measurement sample may be prepared in a buffer solution (e.g., 1 to 5x PBS) in which an immune complex can be formed. Furthermore, during the preparation of the measurement sample, after capturing TDP-43 in the biological sample with the capture antibody, the capture beads may be washed with the buffer solution before binding the detection antibody. Furthermore, after forming an immune complex between TDP-43 in the biological sample, the detection antibody, and the capture antibody on the capture beads, the capture beads may also be washed with the buffer solution.

[0044] The detection antibody may be added to 100 μL of a biological sample or diluted biological sample so that the final concentration is 0.05 to 1 μg / mL (preferably, about 0.1 to 0.5 μg / mL). The detection antibody preferably comprises a labeling substance.

[0045] (Detection Step) In the detection step, a signal derived from the immune complex in the measurement sample is detected.

[0046] Signals derived from immune complexes in a measurement sample can be detected by detecting a labeling substance contained in the immune complex. The labeling substance 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 radioactive isotopes. 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, with β-galactosidase being particularly preferred.

[0047] The enzyme substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when alkaline phosphatase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium) and CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. When β-galactosidase is used as the enzyme, resorufin β-D-galactopyranoside can be used. When alkaline phosphatase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium) and CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. When β-galactosidase is used as the enzyme, resorufin β-D-galactopyranoside can be used. When peroxidase is used as the enzyme, the Super Signal ELISA series from Thermo Fisher Scientific can be used, etc. These enzyme reactions are preferably carried out before placing the beads in the microwells described below, but the enzyme reactions may also be carried out after placing the beads in the microwells.

[0048] Alternatively, the detection antibody may be biotinylated, and the labeling substance may be allowed to bind to labeled avidin or streptavidin, followed by detection of the signal.

[0049] Signal detection can be performed for all or some of the beads, including capture beads and non-capture beads, contained in the measurement sample. Signal detection is preferably performed for each individual bead. Here, since capture beads and non-capture beads differ in the presence or absence, strength, wavelength, etc. of their signals, it is preferable to use a detection device that can distinguish between them.

[0050] For example, when the signal is fluorescent, the signal from each bead may be detected using a flow cytometer. Alternatively, the measurement sample may be brought into contact with a substrate having a plurality of microwells, and the capture beads and non-capture beads in the measurement sample may be placed in the microwells, and the signal in the microwells may be detected.

[0051] The microwells on the substrate can be arranged on a substantially planar surface or in a non-planar three-dimensional array. The microwells can be arranged in a regular pattern or randomly distributed. Preferably, the microwells are in a regular pattern of sites on a substantially planar surface.

[0052] The number of microwells contained in the substrate may be about 1,000 to 1,000,000 per measurement sample, preferably about 10,000 to 500,000, and more preferably about 100,000 to 500,000.

[0053] Substrate materials include, for example, glass (including modified and / or functionalized glass), plastics (such as polypropylene, polyethylene, polybutylene, polyurethane, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), Teflon, polysaccharides, nylon, or nitrocellulose), elastomers (such as dimethylsiloxane and polyurethane), composites, ceramics, silica or silica-based materials (including silicon and modified silicon), carbon, metals, and optical fiber bundles.

[0054] The capacity of each microwell may be set appropriately according to the size of the beads. For example, the number of beads accommodated in one well is approximately 1 to 5, preferably approximately 1 to 2, and more preferably 1. Therefore, the volume of the microwell is preferably large enough to accommodate the above number of beads per well (e.g., U.S. Patent Application Publication No. 2011 / 0212848, WO 2011 / 109364). More specifically, the volume of the microwell is in the range of 100 aL to 1 pL, preferably 1 fL to 500 fL, and more preferably 10 fL to 90 fL. It is preferable that the multiple microwells present on one substrate each have approximately the same volume.

[0055] The method for forming the microwells is not limited as long as it is possible to form microwells of the desired size, and they may be formed by known methods such as photolithography, stamping techniques, molding techniques, and etching techniques.

[0056] Beads can be placed in the microwells using known methods. The beads may be placed in all or some of the microwells on the substrate.

[0057] For example, the measurement sample may be brought into contact with a substrate having microwells, and then the substrate and solution may be centrifuged to place the beads in the microwells. If the beads are magnetic, a magnet may be used to place the beads in the microwells. If the microwells are formed at the end of an optical fiber bundle, the measurement sample may be brought into contact with the other end of the optical fiber, and then centrifugal force, pressure, or other force may be applied to place the beads in the microwells.

[0058] After placing the beads in the microwells, the measurement sample remaining on the substrate around the microwells may be removed, and the surface of the substrate may be washed and / or wiped to remove excess measurement sample. For example, a Simoa™ Disk from Quanterix may be used as a substrate containing microwells.

[0059] After the beads are placed in the microwells, they may be sealed with a fluorous liquid, oil (e.g., mineral oil, fluorinated oil), ferrofluid, non-aqueous polymer solution (e.g., thickener), etc. For example, Simoa™ Sealing Oil from Quanterix may be used for sealing.

[0060] The detection of beads placed in the microwells may be carried out by capturing an image of the signal using a CCD camera or the like when the labeling substance is an enzyme or a fluorescent dye, or by using autoradiography when the labeling substance is a radioisotope.

[0061] The beads placed in the microwells are either capture beads or non-capture beads. Capture beads that have captured TDP-43 emit a signal derived from the labeling substance (i.e., a signal derived from the immune complex), whereas beads that have not captured the target protein and non-capture beads do not emit a signal derived from the labeling substance. Therefore, the target protein can be quantified by counting the number of wells that emit a signal derived from the labeling substance. The microwells from which the signal is detected may be all or some of the microwells on the substrate.

[0062] The number of wells emitting signals derived from the labeled substance can be measured, for example, using the Simoa™ HD-1 Analyzer and Simoa™ HD-X Analyzer from Quanterix. When using the Simoa™ HD-1 Analyzer and Simoa™ HD-X Analyzer, the signal strength is expressed as the average enzymes per bead (AEB) value. Therefore, the AEB value may be used as the signal strength derived from the immune complex in the measurement sample.

[0063] Alternatively, the AEB value may be converted into a measured value of TDP-43 using a calibration curve. Here, the measured value of TDP-43 refers to a value reflecting the amount or concentration of TDP-43. When the measured value is expressed as "amount," it may be in moles or mass, but is preferably expressed as 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 as mass / volume.

[0064] In the estimation method according to one aspect of the present invention, β-galactosidase is preferably used as a labeling substance and resorufin β-D-galactopyranoside is preferably used as a substrate in accordance with the protocol of Quanterix, Inc. Furthermore, it is preferable that the beads are applied to a Simoa™ Disk and the signal intensity is acquired using a Simoa™ HD-1 Analyzer and a Simoa™ HD-X Analyzer.

[0065] A prediction method according to one embodiment of the present invention is a sandwich immunoassay method in which a sandwich immune complex containing TDP-43 is formed and TDP-43 is detected.

[0066] (Estimation Step) In the estimation step, the amount of TDP-43 accumulated in the brain of the subject is estimated based on the intensity of the signal obtained in the detection step. More specifically, the intensity of the signal obtained in the detection step is compared with a predetermined reference value. Then, based on this comparison, the amount of TDP-43 accumulated in the brain is estimated.

[0067] The predetermined reference value refers to a reference value of the signal intensity derived from TDP-43. The reference value may be determined based on the signal intensity derived from TDP-43 in a biological sample from a person who has developed TDP-43 proteinopathy (positive control value).

[0068] Instead of signal intensity, measurements of TDP-43 may be used to estimate the amount of TDP-43 accumulated in the brain of a subject.

[0069] It is known that the TDP-43 concentration in cerebrospinal fluid correlates with the amount of TDP-43 accumulated in the brain. The present inventors have found for the first time that the blood TDP-43 concentration detected using a combination of TDP-43 antibody 1 and TDP-43 antibody 2 correlates with the TDP-43 concentration in cerebrospinal fluid, and that the blood TDP-43 concentration correlates with the amount of TDP-43 accumulated in the brain. Therefore, the amount of TDP-43 accumulated in the brain can be estimated by measuring the TDP-43 concentration in a biological sample using the estimation method according to one embodiment of the present invention.

[0070] [2. Method for Obtaining Information Aiding in Determining Whether or Not a Subject Has a TDP-43 Proteinopathy] A method for obtaining information aiding in determining whether or not a subject has a TDP-43 proteinopathy (hereinafter, may be abbreviated as "information obtaining method") is also included in one aspect of the present invention. The information obtaining method of the present invention obtains information aiding in determining whether or not a subject has a TDP-43 proteinopathy based on the amount of TDP-43 accumulated in the brain of the subject estimated by [1. Method for Estimating the Amount of TDP-43 Accumulated in the Brain of a Subject]. Examples of TDP-43 proteinopathy include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration due to TDP-43 accumulation (FTLD-TDP), limbic-predominant age-related TDP-43 encephalopathy (LATE), and the like.

[0071] More specifically, the estimated amount of TDP-43 accumulated in the subject's brain is compared with a predetermined TDP-43 accumulation reference value. If the estimated amount of TDP-43 accumulated in the subject's brain is higher than the predetermined TDP-43 accumulation reference value, information indicating that the subject is suffering from a TDP-43 proteinopathy is obtained. The predetermined TDP-43 accumulation reference value may be determined based on the amount of TDP-43 accumulated in the brain of a person who has developed a TDP-43 proteinopathy (positive control value). Alternatively, the predetermined TDP-43 accumulation reference value may be a cutoff value determined by receiver operating characteristic curve (ROC) analysis.

[0072] No biomarker for diagnosing LATE before death has been known. By estimating the amount of TDP-43 accumulated in the brain of a subject using this information acquisition method, it is possible to diagnose before death whether the subject is suffering from LATE. Furthermore, LATE can occur alone or in combination with Alzheimer's disease, and it has been revealed that patients with Alzheimer's disease who also have LATE experience rapid progression of cognitive impairment. Determining the presence or absence of LATE pathology is important in the clinical practice and development of treatments for Alzheimer's disease, and this information acquisition method allows appropriate treatment to be administered to the subject.

[0073] [3. Method for Obtaining Information to Aid in Determining Whether or Not a Subject Has FTLD-TDP or an FTLD Other Than FTLD-TDP] One aspect of the present invention also includes a method for obtaining information to aid in determining whether or not a subject has FTLD-TDP or an FTLD other than FTLD-TDP. This method obtains information to aid in determining whether or not a subject has FTLD-TDP or an FTLD other than FTLD-TDP, based on the amount of TDP-43 accumulated in the brain of a subject estimated by [1. Method for Estimating the Amount of TDP-43 Accumulated in the Brain of a Subject]. Examples of FTLD other than FTLD-TDP include frontotemporal lobar degeneration caused by tau accumulation (FTLD-tau), frontotemporal lobar degeneration caused by FUS (fused in sarcoma) accumulation (FTLD-FUS), and frontotemporal lobar degeneration caused by UPS (ubiquitin proteasome system) accumulation (FTLD-UPS).

[0074] In a method for obtaining information to assist in determining whether a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, more specifically, the estimated amount of TDP-43 accumulated in the brain of the subject is compared with a predetermined TDP-43 accumulation reference value. Then, when the estimated amount of TDP-43 accumulated in the brain of the subject is higher than the predetermined TDP-43 accumulation reference value, information that the subject is suffering from FTLD-TDP is obtained. The predetermined TDP-43 accumulation reference value may be determined based on the amount of TDP-43 accumulated in the brain of a person suffering from FTLD-TDP (positive control value).

[0075] The predetermined reference value for TDP-43 accumulation may be a cutoff value determined by ROC (receiver operating characteristic curve) analysis.

[0076] The treatment methods for FTLD-TDP and FTLDs other than FTLD-TDP (for example, FTLD-tau) are fundamentally different. The "method for obtaining information that assists in determining whether a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP" enables appropriate treatment to be administered depending on the pathology suffered by the subject.

[0077] 4. Apparatus for Estimating TDP-43 Accumulation in the Brain of a Subject The present invention also relates to an apparatus for estimating TDP-43 accumulation in the brain of a subject (hereinafter, may be abbreviated as "estimation apparatus"). An estimation apparatus according to one embodiment of the present invention will be described in detail with reference to FIG. 3. FIG. 3 is a diagram showing a schematic configuration of an estimation apparatus 20 according to one embodiment of the present invention. The estimation apparatus 20 is an apparatus that estimates TDP-43 accumulation in the brain of a subject by detecting TDP-43 in a biological sample collected from the subject.

[0078] 3 , the estimation device 20 is connected to an input unit 30, an output unit 31, and a storage medium 32. The estimation device 20 includes a processing unit (CPU) 21, a main storage unit 22, a ROM (read only memory) 23, an auxiliary storage unit 24, a communication interface (I / F) 28, an input interface (I / F) 25, an output interface (I / F) 26, and a media interface (I / F) 27, which are connected to each other via a bus 29 so as to be able to communicate data with each other.

[0079] The CPU 21 is a processing unit of the estimation 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 estimation device 20 to function.

[0080] 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 therein. The CPU 21 may be an MPU 21. The ROM 23 stores a boot program executed by the CPU 21 when the estimation device 20 is started up, as well as programs and settings related to the operation of the hardware of the estimation device 20.

[0081] 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.

[0082] The auxiliary storage unit 24 is configured by a hard disk, a semiconductor memory element 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 measurement programs, reference values ​​for signal strength, reference values ​​for TDP-43 measurement values, etc., which will be described later.

[0083] The communication I / F 28 is composed of a serial interface such as USB, IEEE 1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE 1284, an analog interface including a D / A converter, an A / D converter, 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 estimation 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.

[0084] The input I / F 25 is composed of, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter and an A / D converter. The input I / F 25 has an input unit 30 that 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.

[0085] 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 estimation device 20. The input unit 30 may be connected to the estimation device 20 from outside, or may be integrated with the estimation device 20.

[0086] 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.

[0087] 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 estimation device 20, measurement results, etc.

[0088] 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.

[0089] The storage medium 32 is configured by 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.

[0090] The CPU 21 may acquire application software and various settings necessary for controlling the estimation 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 estimation 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.

[0091] Furthermore, the ROM 23 or the auxiliary storage unit 24 may have installed therein 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.

[0092] The processing unit 21 receives input from the input unit 30 by the examiner to acquire signals derived from immune complexes for a measurement sample containing non-capture beads and capture beads bound to immune complexes. The processing unit 21 then acquires signals derived from immune complexes detected by a measurement unit or the like (described below). The processing unit 21 then stores the intensities of these acquired signals in the auxiliary memory unit 24 or the like.

[0093] The estimation device 20 may be a personal computer, etc. The estimation device 20 may also be built into a measurement unit, which will be described later.

[0094] 5. System for Estimating the Amount of TDP-43 Accumulated in a Subject's Brain The present invention also relates to a system for estimating the amount of TDP-43 accumulated in a subject's brain. The system for estimating the amount of TDP-43 accumulated in a subject's brain includes an estimation device 20 and a measurement unit. The system for estimating the amount of TDP-43 accumulated in a subject's brain detects TDP-43 in a biological sample collected from the subject and estimates the amount of TDP-43 accumulated in the subject's brain.

[0095] The measurement unit includes a detection unit having an imaging device such as a CCD camera for detecting signals, and may further include a sample preparation unit for preparing a measurement sample. The detection unit may also be a flow cytometer. The detection unit may also include a pressure device, a pressure reduction device, or a centrifugal device for placing beads in the measurement sample into the microwells.

[0096] The measurement unit may be integrated with the estimation device 20. Examples of devices that include an imaging device in the detection unit and are integrated with the estimation device 20 include the Simoa (trademark) HD-1 Analyzer and the Simoa (trademark) HD-X Analyzer.

[0097] 6. Device for Acquiring Information to Assist in Determining Whether or Not a Subject Has a TDP-43 Proteinopathy A device for acquiring information to assist in determining whether or not a subject has a TDP-43 proteinopathy (hereinafter, sometimes abbreviated as "information acquisition device") is also included in one aspect of the present invention. An information acquisition device according to one embodiment of the present invention is shown in FIG. 4. Information acquisition device 50 is a device that acquires information as to whether or not a subject has a TDP-43 proteinopathy, based on the amount of TDP-43 accumulated in the brain of the subject, which is estimated by detecting TDP-43 in a biological sample collected from the subject.

[0098] 4, 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 storage unit 52, ROM (read only memory) 53, auxiliary storage 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.

[0099] The details of each component in FIG. 4 are the same as the details of each component in FIG.

[0100] The processing unit 51 accepts input from the tester via the input unit 60 to acquire signals derived from immune complexes from a measurement sample containing non-capture beads and capture beads bound to immune complexes. Next, signals derived from the immune complexes detected by a measurement unit or the like, described below, are acquired. The acquired signal intensity is then compared with a reference value for signal intensity stored in the auxiliary memory unit 54 or the like. Based on this comparison, the processing unit 51 estimates the amount of TDP-43 accumulated in the brain. If the estimated amount of TDP-43 accumulated in the brain is lower than the TDP-43 accumulation reference value, the processing unit 51 determines that the subject is unlikely to be suffering from a TDP-43 proteinopathy. The processing unit 51 stores these determination results in the auxiliary memory unit 54 or the like as information on whether or not the subject has a TDP-43 proteinopathy. The processing unit 51 may output these results to the output unit 61 or store them in a storage medium 62.

[0101] [7. System for Acquiring Information to Aid in Determining Whether or Not a Subject Has a TDP-43 Proteinopathy] A system for acquiring information to aid in determining whether or not a subject has a TDP-43 proteinopathy (hereinafter, sometimes abbreviated as "information acquisition system") is also included in one aspect of the present invention. The information acquisition system includes an information acquisition device 50 and a measurement unit. The information acquisition system is a system for acquiring information to aid in determining whether or not a subject has a TDP-43 proteinopathy based on an estimated amount of TDP-43 accumulated in the brain of the subject. Details of the measurement unit are the same as those of the measurement unit described in [4. System for Estimating the Amount of TDP-43 Accumulated in the Brain of a Subject].

[0102] The measurement unit may be integrated with the information acquisition device 50. Examples of devices that include an imaging device in the detection unit and are integrated with the information acquisition device 50 include the Simoa (trademark) HD-1 Analyzer and the Simoa (trademark) HD-X Analyzer.

[0103] 8. Device or System for Acquiring Information to Aid in Determining Whether a Subject Has FTLD-TDP or an FTLD Other Than FTLD-TDP] One aspect of the present invention also includes a device for acquiring information to aid in determining whether a subject has FTLD-TDP or an FTLD other than FTLD-TDP. The configuration of the device may be the same as the configuration of the device for acquiring information to aid in determining whether a subject has a TDP-43 proteinopathy, as shown in Figure 4. When the device has the configuration of Figure 4, the processing unit 51 estimates the amount of TDP-43 accumulated in the brain, and if the estimated amount of TDP-43 accumulated in the brain is higher than the reference amount of TDP-43 accumulation, the processing unit 51 determines that the subject has FTLD-TDP.

[0104] Another aspect of the present invention also includes a system for acquiring information that aids in determining whether or not a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP. This system acquires information that aids in determining whether or not a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on an estimated amount of TDP-43 accumulated in the brain of the subject. The configuration of this system may be the same as that of a system for acquiring information that aids in determining whether or not a subject is suffering from a TDP-43 proteinopathy.

[0105] 9. Computer Program for Estimating the Amount of TDP-43 Accumulated in a Subject's Brain The present invention also relates to a computer program (hereinafter sometimes abbreviated as "estimation program") for estimating the amount of TDP-43 accumulated in a subject's brain. The estimation program is used as a measurement program that controls the operation of the estimation device 20. The estimation program causes a computer to execute the steps of acquiring a signal derived from an immune complex for a measurement sample containing non-capture beads and capture beads to which an immune complex is bound, and estimating the amount of TDP-43 accumulated in the subject's brain based on the intensity of the signal.

[0106] Furthermore, the measurement program and the information acquisition program may be stored in a storage medium such as a hard disk, a semiconductor memory element such as a flash memory, an optical disk, etc. The format of the program stored in the storage medium is not limited as long as the information acquisition device can read the program. It is preferable that the storage medium is non-volatile.

[0107] 10. Computer Program for Acquiring Information to Assist in Determining Whether or Not a Subject Has a TDP-43 Proteinopathy Another aspect of the present invention includes a computer program (hereinafter sometimes abbreviated as "information acquisition program") for acquiring information to assist in determining whether or not a subject has a TDP-43 proteinopathy. 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 derived from an immune complex for a measurement sample containing non-capture beads and capture beads to which an immune complex is bound. The information acquisition program then causes the computer to execute a step of acquiring information as to whether or not the subject from whom the biological sample was collected has a TDP-43 proteinopathy, based on the estimated amount of TDP-43 accumulated in the subject's brain.

[0108] [11. Computer Program for Acquiring Information Aiding in the Determination of Whether or Not a Subject Has FTLD-TDP or an FTLD Other Than FTLD-TDP] One aspect of the present invention also includes a computer program for acquiring information that aids in the determination of whether or not a subject has FTLD-TDP or an FTLD other than FTLD-TDP. The program is used as a measurement program that controls the operation of an apparatus for acquiring information that aids in the determination of whether or not a subject has FTLD-TDP or an FTLD other than FTLD-TDP. The information acquisition program causes a computer to execute a step of acquiring a signal derived from an immune complex for a measurement sample containing non-capture beads and capture beads to which an immune complex is bound. The information acquisition program then causes the computer to execute a step of acquiring information as to whether or not the subject from whom the biological sample was collected has FTLD-TDP or an FTLD other than FTLD-TDP, based on the estimated amount of TDP-43 accumulated in the subject's brain.

[0109] [12. TDP-43 Detection Reagent and Kit Comprising the Detection Reagent] A reagent for detecting TDP-43 (hereinafter sometimes abbreviated as "detection reagent") is also included in one aspect of the present invention. The detection reagent according to one aspect of the present invention is used in [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject] and comprises the capture beads and non-capture beads. The preferred ratio of the number of capture beads to non-capture beads contained in the detection reagent is as described in [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject].

[0110] A detection reagent according to one embodiment of the present invention is a reagent for estimating the amount of TDP-43 accumulated in the brain of a subject. The reagent includes a detection antibody and a capture antibody. One of the detection antibody and the capture antibody is TDP-43 antibody 1. The other of the detection antibody and the capture antibody is TDP-43 antibody 2 or a phosphorylated TDP-43 antibody.

[0111] A reagent kit for detecting TDP-43 (hereinafter, sometimes abbreviated as "reagent kit") is also included in one aspect of the present invention. The reagent kit according to one aspect of the present invention is used in [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject] and includes the above-mentioned detection reagent. Furthermore, the reagent kit according to one aspect of the present invention is a reagent kit for estimating the amount of TDP-43 accumulated in the brain of a subject and includes the above-mentioned detection reagent.

[0112] The reagent kit may further include a detection antibody, and may also include other reagents and tools, such as a blocking agent such as albumin, casein, or skim milk, and a buffer solution (which may contain the above-mentioned blocking agent and EDTA) used to suspend the beads and dilute the sample. If the capture antibody is not immobilized on the beads, the reagent kit may further include a reagent containing a capture antibody. The reagent kit may also include a mixture of multiple different reagents 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 TDP-43 measurement procedure, etc. These 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.

[0113] Another aspect of the present invention is a kit for estimating the amount of TDP-43 accumulated in the brain of a subject, which includes the above-mentioned detection reagent. The kit may also include a computer-readable recording medium storing a computer program for estimating the amount of TDP-43 accumulated in the brain of a subject. Examples of recording media include hard disks, semiconductor memory devices such as flash memories, and optical disks.

[0114] Another aspect of the present invention is a kit for obtaining information that aids in determining whether or not a person has a TDP-43 proteinopathy, which includes the above-mentioned detection reagent. The kit may also include a computer-readable recording medium that stores a computer program for obtaining information that aids in determining whether or not a person has a TDP-43 proteinopathy.

[0115] Another aspect of the present invention is a kit for obtaining information that aids in determining whether or not a person has FTLD-TDP or an FTLD other than FTLD-TDP, the kit including the above-mentioned detection reagent. The kit may also include a computer-readable recording medium that stores a computer program for obtaining information that aids in determining whether or not a person has FTLD-TDP or an FTLD other than FTLD-TDP.

[0116] [13. Method for diagnosing TDP-43 proteinopathy] A method for diagnosing TDP-43 proteinopathy is also included in one aspect of the present invention. The diagnostic method includes a diagnostic step of diagnosing whether or not a subject suffers from a TDP-43 proteinopathy based on the amount of TDP-43 accumulated in the brain estimated by [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject]. The diagnostic method encompasses diagnosis by a physician or other qualified medical professional.

[0117] In the above diagnostic step, the estimated amount of TDP-43 accumulation in the brain is compared with a predetermined TDP-43 accumulation reference value. If the estimated amount of TDP-43 accumulation in the brain is higher than the predetermined TDP-43 accumulation reference value, the subject is diagnosed as having a TDP-43 proteinopathy. The "TDP-43 accumulation reference value" is as described in [2. Method for obtaining information to assist in determining whether or not a subject has a TDP-43 proteinopathy].

[0118] Instead of signal intensity, TDP-43 measurements may be used to estimate the amount of TDP-43 accumulated in the brain of a subject and to diagnose whether the subject is suffering from a TDP-43 proteinopathy.

[0119] By diagnosing whether or not a subject is suffering from TDP-43 proteinopathy, it is possible to objectively observe and evaluate the progression of the disease, the progress of treatment, drug efficacy evaluation (new drug development), or the effect of treatment.

[0120] [14. Method for diagnosing FTLD pathology] One aspect of the present invention also includes a method for diagnosing FTLD pathology. The diagnostic method includes a diagnostic step of diagnosing whether a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the amount of TDP-43 accumulated in the brain estimated by [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject]. The diagnostic method encompasses diagnosis by a physician or other qualified medical professional.

[0121] In the above diagnostic step, the estimated amount of TDP-43 accumulation in the brain is compared with a predetermined TDP-43 accumulation reference value. If the estimated amount of TDP-43 accumulation in the brain is higher than the predetermined TDP-43 accumulation reference value, the subject is diagnosed as having FTLD-TDP. The "TDP-43 accumulation reference value" is as described in [3. Method for obtaining information to assist in determining whether a subject has FTLD-TDP or an FTLD other than FTLD-TDP].

[0122] Instead of signal intensity, the measured value of TDP-43 may be used to estimate the amount of TDP-43 accumulated in the brain of a subject and to diagnose whether the subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP.

[0123] By diagnosing the pathology of FTLD in a subject, it is possible to provide a treatment method appropriate for the subject depending on the pathology.

[0124] [15. Method for Assisting Patient Diagnosis] A method for assisting patient diagnosis is also included in one embodiment of the present invention. One embodiment of the method for assisting patient diagnosis comprises a step of obtaining data suggesting whether or not a biological sample is derived from a patient suffering from a TDP-43 proteinopathy, based on the amount of TDP-43 accumulated in the patient's brain estimated by [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject]. Another embodiment of the method for assisting patient diagnosis comprises a step of obtaining data suggesting whether or not a biological sample is derived from a patient suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the amount of TDP-43 accumulated in the patient's brain estimated by [1. Method for estimating the amount of TDP-43 accumulated in the brain of a subject].

[0125] 16. Summary of Embodiments The embodiments of the present invention include, for example, the following aspects. <1> A method for estimating the amount of TDP-43 accumulated in the brain of a subject, comprising: a preparation step of forming an immune complex between TDP-43 in a biological sample collected from the subject, a capture antibody, and a detection antibody on capture beads in the presence of non-capture beads, thereby preparing a measurement sample containing the non-capture beads and capture beads to which the immune complex is bound; a detection step of detecting a signal derived from the immune complex in the measurement sample; and an estimation step of estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, wherein the non-capture beads do not bind to the immune complex, one of the epitope of the detection antibody and the capture antibody comprises a peptide consisting of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated. <2> The method according to <1>, wherein the TDP-43 is phosphorylated TDP-43. <3> The method according to <1> or <2>, wherein, in the detection step, the measurement sample is brought into contact with a substrate having a plurality of microwells, capture beads and non-capture beads in the measurement sample are placed in the microwells one by one, and a signal derived from an immune complex on the capture bead placed in the microwell is detected, wherein the microwell has a volume capable of accommodating only one capture bead or non-capture bead. <4> The method according to any one of <1> to <3>, wherein, in the detection step, the signal is detected by imaging the signal derived from the immune complex. <5> The method according to any one of <1> to <4>, wherein the biological sample is a blood sample. <6> The method according to <5>, wherein the blood sample is serum or plasma. <7> The method according to any one of <1> to <4>, wherein the biological sample is a tissue extract sample. <8> The method according to any one of <1> to <7>, wherein the immune complex is a complex of a capture antibody, TDP-43 in a biological sample, and a detection antibody, and the capture antibody binds to a capture bead.<9> The method according to any one of <1> to <8>, wherein the immune complex is formed by mixing a capture antibody bound to capture beads, a biological sample containing TDP-43, and a detection antibody in the presence of non-capture beads. <10> A method for obtaining information that aids in determining whether a subject is suffering from a TDP-43 proteinopathy, based on the amount of TDP-43 accumulated in the brain estimated by the method according to any one of <1> to <9>. <11> A method for obtaining information that aids in determining whether a subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP, based on the amount of TDP-43 accumulated in the brain estimated by the method according to any one of <1> to <9>. <12> A reagent or reagent kit for use in the method according to any one of <1> to <9>, comprising the capture beads and the non-capture beads. <13> An apparatus for estimating the amount of TDP-43 accumulated in the brain of a subject, the apparatus comprising a processing unit, the processing unit being configured to form an immune complex between TDP-43 in a biological sample collected from a subject, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, for a measurement sample containing the non-capture beads and the capture beads to which the immune complex is bound, and acquiring a signal derived from the immune complex, and estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, the non-capture beads not binding to the immune complex, one epitope of the detection antibody and the capture antibody comprising a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the other epitope of the detection antibody and the capture antibody comprising an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated.<14> An apparatus for obtaining information on whether a subject suffers from a TDP-43 proteinopathy based on the intensity of a signal derived from TDP-43 in a biological sample collected from the subject, the apparatus comprising a processing unit, wherein the processing unit obtains a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads to which the immune complex is bound, the measurement sample being prepared by forming an immune complex between TDP-43 in the biological sample collected from the subject, a detection antibody, and a capture antibody on the non-capture beads in the presence of the non-capture beads, and estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, and estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal. The device obtains information on whether the subject is suffering from TDP-43 proteinopathy based on the amount of accumulated TDP-43, wherein the non-capture beads do not bind to the immune complex, the epitope of one of the detection antibody and capture antibody comprises a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated.<15> An apparatus for obtaining information on whether a subject is affected with FTLD-TDP or an FTLD other than FTLD-TDP, based on the intensity of a signal derived from TDP-43 in a biological sample collected from the subject, the apparatus comprising a processing unit, which acquires a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads to which the immune complex is bound, the measurement sample being prepared by forming an immune complex between TDP-43 in the biological sample collected from the subject, a detection antibody, and a capture antibody on the non-capture beads in the presence of the non-capture beads, and estimates the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, and estimates the amount of TDP-43 accumulated in the brain of the subject based on the estimated amount of TDP-43. The device obtains information on whether the subject is suffering from FTLD-TDP or an FTLD other than FTLD-TDP based on the amount of TDP-43 accumulated in the brain, wherein the non-capture beads do not bind to the immune complex, the epitope of one of the detection antibody and capture antibody comprises a peptide of amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope comprising a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated.<16> A step of acquiring a signal derived from the immune complex for a measurement sample containing non-capture beads and capture beads bound with the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the biological sample is collected from a subject, the non-capture beads do not bind to the immune complex, and the epitope of one of the detection antibody and the capture antibody is located between amino acids 203 and 209 of the amino acid sequence shown in SEQ ID NO: 1. wherein the epitope of the other of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serines 409 and 410 are phosphorylated; and estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal.<17> A step of acquiring a signal derived from an immune complex for a measurement sample containing non-capture beads and capture beads bound with the immune complex, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the non-capture beads are collected from a subject, the non-capture beads do not bind to the immune complex, one epitope of the detection antibody and the capture antibody comprises a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the other epitope of the detection antibody and the capture antibody comprises a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1. the signal intensity is an epitope contained in a peptide consisting of amino acids 261 to 414 of a sequence, or an epitope containing a peptide consisting of amino acids 404 to 410 of a sequence in which serines 409 and 410 are phosphorylated; estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal; and obtaining information on whether the subject from whom the biological sample was collected is affected with TDP-43 proteinopathy based on the estimated amount of TDP-43 accumulated in the brain of the subject.<18> A step of acquiring a signal derived from an immune complex for a measurement sample containing non-capture beads and capture beads to which the immune complex is bound, the measurement sample being prepared by forming an immune complex between TDP-43 in a biological sample, a detection antibody, and a capture antibody on the capture beads in the presence of non-capture beads, wherein the non-capture beads are not bound to the immune complex, and the epitope of one of the detection antibody and the capture antibody comprises a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO: 1, and the epitope of the other of the detection antibody and the capture antibody comprises a peptide from amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1. or an epitope comprising a peptide consisting of amino acids 404 to 410, in which serines 409 and 410 are phosphorylated, estimating the amount of TDP-43 accumulated in the brain of the subject based on the intensity of the signal, and obtaining information on whether the subject from whom the biological sample was collected is affected with FTLD-TDP or an FTLD other than FTLD-TDP based on the estimated amount of TDP-43 accumulated in the brain of the subject. <19> A method for diagnosing TDP-43 proteinopathy, comprising a diagnostic step of diagnosing whether a subject is affected with TDP-43 proteinopathy based on the amount of TDP-43 accumulated in the brain estimated by the method described in any of <1> to <9>. <20> A method for assisting in the diagnosis of a patient, comprising a step of obtaining data suggesting whether a biological sample is derived from a patient suffering from a TDP-43 proteinopathy, based on the amount of accumulated TDP-43 in the brain estimated by the method described in any one of <1> to <9>. <21> A method for diagnosing the pathology of FTLD, comprising a diagnostic step of diagnosing whether a subject suffers from FTLD-TDP or an FTLD other than FTLD-TDP, based on the amount of accumulated TDP-43 in the brain estimated by the method described in any one of <1> to <9>.<20> A method for assisting in the diagnosis of a patient, comprising a step of obtaining data indicating whether a biological sample is derived from a patient suffering from FTLD-TDP, based on the amount of accumulated TDP-43 in the brain estimated by the method according to any one of <1> to <9>.

[0126] 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.

[0127] [Materials] 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. 60019-2-Ig (Proteintech) was used as the capture antibody. The antigen peptide of 60019-2-Ig corresponds to amino acid residues 203-209 of the amino acid sequence of SEQ ID NO: 1. 12892-1-AP (Proteintech) was used as the detection antibody, and biotinylation of the detection antibody was prepared according to the kit's protocol. The antigen peptide of 12892-1-AP is contained in the peptide consisting of amino acid residues 261-414 of the amino acid sequence of SEQ ID NO: 1. 12892-1-AP is a polyclonal antibody. Hereinafter, the method for measuring TDP-43 using a combination of 60019-2-Ig (capture antibody) and 12892-1-AP (detection antibody) may be abbreviated as the measurement method of the Examples.

[0128] Non-phosphorylated TDP-43 was used as the target protein. A human TDP-43 standard peptide was used as a standard peptide to generate a standard curve. The target protein or standard peptide was diluted using the sample diluent in the Simoa™ Homebrew Assay Development kit.

[0129] [Evaluation Example 1] Twenty FTLD patients were imaged using a tau PET system using PM-PBB3 as a tau PET ligand at the National Institutes for Quantum and Radiological Science and Technology. Results indicated 16 Tau-PET-positive patients (patients with FTLD-tau) and 4 Tau-PET-negative patients (patients with FTLD-TDP). Plasma TDP-43 concentrations were measured for the 20 FTLD patients, and blood TDP-43 concentrations were compared. Plasma was diluted 4-fold with Sample Diluent. Buffer was also used as a blank. Plasma TDP-43 concentrations were measured.

[0130] Measurements were performed using the Simoa™ Enzyme and Substrate kit, Simoa™ Disk kit, Simoa™ Sealing Oil, System buffer 1, System buffer 2, and the Simoa™ HD-1 Analyzer according to the manufacturer's protocol. The Simoa™ HD-1 Analyzer was loaded with the measurement sample and necessary reagents, including capture beads, biotin-labeled antibodies, and non-capture beads. All measurement steps were then performed using the Simoa™ HD-1 Analyzer.

[0131] In Evaluation Example 1, the ratio of the number of capture beads to non-capture beads was adjusted so that the number of non-capture beads was 3 to 9 for every 1 capture bead. The concentration of all beads (capture beads and non-capture beads) was adjusted to 400,000 to 500,000 beads per 90 μL of measurement sample.

[0132] The specific measurement process is as follows: First, the capture antibody bound to the capture beads was allowed to bind to a standard protein in the blood in the presence of non-capture beads. Next, a biotinylated detection antibody was allowed to further bind to the standard protein bound to the capture antibody, forming an immune complex of the capture antibody, standard protein, and biotinylated detection antibody on the capture beads.

[0133] After immune complex formation, the capture beads were reacted with β-galactosidase-conjugated streptavidin in the presence of non-capture beads. The capture beads were then reacted with a β-galactosidase substrate (RGP: resorufin β-D-galactopyranoside) in the presence of non-capture beads. After the RGP reaction, the capture beads and non-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 (fL) wells. Each bead in the well was photographed using a Simoa™ HD-1 Analyzer, and its fluorescence intensity (AEB) was measured. The target protein was quantified by counting the number of beads with high fluorescence intensity.

[0134] <Results of Evaluation Example 1> The results of Evaluation Example 1 are shown in Figure 1. As shown in Figure 1, the blood TDP-43 concentrations measured by the measurement method of the Example were statistically significantly higher in FTLD-TDP patients compared to FTLD-tau patients. These results demonstrate that the blood TDP-43 concentrations measured by the measurement method of the Example reflect the actual amount of TDP-43 accumulated in the patient's brain. Furthermore, it was found that the blood TDP-43 concentrations measured by the measurement method of the Example enable differential diagnosis of whether a patient is suffering from FTLD-TDP or FTLD-tau.

[0135] [Evaluation Example 2] Verification of the feasibility of distinguishing between FTLD-tau and FTLD-TDP using the GFAP / NfL ratio Non-Patent Document 3 discloses that FTLD-tau and FTLD-TDP can be distinguished from each other by the ratio of glial fibrillary acidic protein (GFAP) to neurofilament light chain (NfL) in the blood. Whether this disclosure is correct was verified in this evaluation example.

[0136] For 16 Tau-PET-positive patients and 4 Tau-PET-negative patients (patients suffering from FTLD-TDP) whose blood TDP-43 concentrations were measured in Evaluation Example 1, plasma NfL concentrations and plasma GFAP concentrations were measured according to the method disclosed in Non-Patent Document 3, and the GFAP / NfL ratio was calculated. The results are shown in Figure 2. In Figure 2, "HC" indicates normal subjects (healthy control).

[0137] As shown in Figure 2, the GFAP / NfL ratio in the blood could not distinguish between actual FTLD-tau patients and FTLD-TDP patients. Non-Patent Document 3 also excluded patients with Alzheimer's disease (AD) pathology from the study, but since GFAP and NfL levels increase in the presence of AD pathology, differentiation based on the GFAP / NfL ratio is not possible in elderly people who frequently have AD pathology (e.g., 25% of 70-year-olds have amyloid accumulation even with normal cognitive function). The results in Figure 2 also demonstrate this.

[0138] The method of the present invention can provide information that can be used for highly sensitive biochemical diagnosis of TDP-43 proteinopathies such as FTLD-TDP, and can be used for, for example, pharmaceutical purposes.

[0139] 20 Measuring device 21, 51 Processing unit (CPU) 22, 52 Main memory unit 23, 53 ROM 24, 54 Auxiliary memory unit 25, 55 Input I / F 26, 56 Output I / F 27, 57 Media I / F 28, 58 Communication I / F 29, 59 Bus 30, 60 Input unit 31, 61 Output unit 32, 62 Storage medium 50 Information input device

Claims

1. Preparation step of preparing a measurement sample including the non-captured beads and the captured beads to which the immune complex is bound, by forming an immune complex on the captured beads with TDP-43 from a biological sample taken from a subject, a captured antibody, and a detection antibody in the presence of non-captured beads. A detection step for detecting a signal originating from an immune complex in the sample being measured, and The estimation step includes estimating the amount of TDP-43 accumulated in the subject's brain based on the intensity of the signal, The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. A method for estimating the amount of TDP-43 accumulated in a subject's brain, wherein the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated.

2. The method according to claim 1, wherein TDP-43 is phosphorylated TDP-43.

3. In the detection step, The sample to be measured is brought into contact with a substrate having multiple microwells. The captured beads and uncaptured beads from the measurement sample are placed one bead each in the microwells. The signal originating from the immune complex on the capture beads arranged in the microwell is detected, The method according to claim 1, wherein the microwell has a volume capable of accommodating only one of the captured beads or uncaptured beads.

4. The method according to claim 1, wherein the detection step is performed by imaging the signal originating from the immune complex.

5. The method according to claim 1, wherein the biological sample is a blood sample.

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

7. The method according to claim 1, wherein the biological sample is a tissue extract sample.

8. The method according to claim 1, wherein the immune complex is a complex of a capture antibody, TDP-43 in a biological sample, and a detection antibody, and the capture antibody is bound to a capture bead.

9. The method according to claim 1, wherein the immune complex is formed by mixing a capture antibody bound to a capture bead, a biological sample containing TDP-43, and a detection antibody in the presence of non-capture beads.

10. A method for obtaining information to assist in determining whether a subject has TDP-43 proteinopathy, based on the amount of TDP-43 accumulation in the brain estimated by the method according to any one of claims 1 to 9.

11. A method for obtaining information to assist in determining whether a subject is suffering from FTLD-TDP or FTLD other than FTLD-TDP, based on the amount of TDP-43 accumulation in the brain estimated by the method according to any one of claims 1 to 9.

12. A reagent or reagent kit used in the method according to any one of claims 1 to 9, comprising the capture beads and the non-capture beads.

13. Equipped with a processing unit, The aforementioned processing unit, In the presence of non-capture beads, an immune complex is prepared by forming an immune complex on capture beads with TDP-43 from a biological sample taken from a subject, a detection antibody, and a capture antibody, and the measurement sample includes the non-capture beads and the capture beads to which the immune complex is bound, and a signal originating from the immune complex is obtained. Based on the intensity of the signal, the amount of TDP-43 accumulated in the subject's brain is estimated. The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. A device for estimating the amount of TDP-43 accumulated in a subject's brain, wherein the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated.

14. A device for obtaining information on whether or not a subject suffers from TDP-43 proteinopathy, based on the intensity of the signal derived from TDP-43 in a biological sample taken from the subject, Equipped with a processing unit, The aforementioned processing unit, In the presence of non-capture beads, an immune complex is prepared by forming an immune complex on capture beads with TDP-43 from a biological sample taken from a subject, a detection antibody, and a capture antibody, and the measurement sample includes the non-capture beads and the capture beads to which the immune complex is bound, and a signal originating from the immune complex is obtained. Based on the intensity of the signal, the amount of TDP-43 accumulated in the subject's brain is estimated. Based on the estimated amount of TDP-43 accumulation in the subject's brain, information is obtained as to whether the subject suffers from TDP-43 proteinopathy. The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. The apparatus wherein the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated.

15. A device for acquiring information on whether a subject is suffering from FTLD-TDP or FTLD other than FTLD-TDP, based on the intensity of the signal derived from TDP-43 in a biological sample taken from the subject, The device comprises a processing unit, The aforementioned processing unit, In the presence of non-capture beads, an immune complex is prepared by forming an immune complex on capture beads with TDP-43 from a biological sample taken from a subject, a detection antibody, and a capture antibody, and the measurement sample includes the non-capture beads and the capture beads to which the immune complex is bound, and a signal originating from the immune complex is obtained. Based on the intensity of the signal, the amount of TDP-43 accumulated in the subject's brain is estimated. Based on the estimated amount of TDP-43 accumulated in the subject's brain, information is obtained as to whether the subject has FTLD-TDP or FTLD other than FTLD-TDP. The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. The apparatus wherein the other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated.

16. On the computer, A step of obtaining a signal derived from the immune complex with respect to a measurement sample that includes the non-captured beads and the captured beads to which the immune complex is bound, prepared by forming an immune complex of TDP-43 in a biological sample, a detection antibody, and a capture antibody on captured beads in the presence of non-captured beads, The aforementioned biological sample was taken from a subject, The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. The other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated. A step of estimating the amount of TDP-43 accumulated in the subject's brain based on the intensity of the signal, A computer program that runs to estimate the amount of TDP-43 accumulated in the subject's brain.

17. On the computer, A step of obtaining a signal derived from the immune complex with respect to a measurement sample that includes the non-captured beads and the captured beads to which the immune complex is bound, prepared by forming an immune complex of TDP-43 in a biological sample, a detection antibody, and a capture antibody on captured beads in the presence of non-captured beads, The aforementioned biological sample was taken from a subject, The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. The other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated. A step of estimating the amount of TDP-43 accumulated in the subject's brain based on the intensity of the signal, Based on the estimated amount of TDP-43 accumulated in the subject's brain, the step of obtaining information on whether or not the subject from whom the biological sample was taken suffers from TDP-43 proteinopathy, A computer program that executes a command to obtain information on whether or not a person has TDP-43 proteinopathy.

18. On the computer, A step of obtaining a signal derived from the immune complex with respect to a measurement sample that includes the non-captured beads and the captured beads to which the immune complex is bound, prepared by forming an immune complex of TDP-43 in a biological sample, a detection antibody, and a capture antibody on captured beads in the presence of non-captured beads, The aforementioned biological sample was taken from a subject, The non-captured beads do not bind to the immune complex. One of the epitopes of the detection antibody and the capture antibody contains a peptide from amino acids 203 to 209 of the amino acid sequence shown in SEQ ID NO:

1. The other epitope of the detection antibody and the capture antibody is an epitope contained in a peptide consisting of amino acids 261 to 414 of the amino acid sequence shown in SEQ ID NO: 1, or an epitope containing a peptide consisting of amino acids 404 to 410 in which serine at positions 409 and 410 is phosphorylated. A step of estimating the amount of TDP-43 accumulated in the subject's brain based on the intensity of the signal, Based on the estimated amount of TDP-43 accumulated in the subject's brain, the step of obtaining information on whether the subject from whom the biological sample was taken is suffering from FTLD-TDP or FTLD other than FTLD-TDP, A computer program that executes the following to obtain information on whether or not a person has FTLD-TDP or another type of FTLD.