Method for testing alzheimer's disease and test kit for alzheimer's disease

Abstract

To provide a new method usable for examination of Alzheimer's disease.SOLUTION: A method for testing Alzheimer's disease according to the present invention includes: a measurement step of measuring an amount of a target phosphorylated portion in two or more types of peptide fragments derived from a phosphorylated tau protein in a sample of a subject using two or more measurement systems; and a comparison step of comparing the magnitudes of the measured values of the target phosphorylation portion. The measurement step includes: a first measurement step of measuring the amount of the target phosphorylated portion using a first measurement system including a first binding molecule that binds to the target phosphorylated portion and a second binding molecule that binds to a non-phosphorylated portion of the peptide fragment; and a second measurement step of measuring the amount of the target phosphorylated portion using a second measurement system including a third binding molecule that binds to the target phosphorylated portion and a fourth binding molecule that binds to a non-phosphorylated portion of the peptide fragment.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for testing for Alzheimer's disease and a test kit for Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease, like lifestyle-related diseases, is considered to be a continuous disease. It is known that the accumulation of amyloid beta in the brain from an asymptomatic stage, followed by an increase in tau protein (tau), and neurofibrillary tangles due to tau phosphorylation and aggregation, leads to neuronal damage and cognitive impairment (Non-Patent Document 1).

[0003] An anti-amyloid beta (Aβ) antibody has been approved in Japan as a disease-modifying drug for Alzheimer's disease (AD). The approval requires that the Aβ42 / Aβ40 ratio in cerebrospinal fluid be measured as a body fluid biomarker to confirm amyloid accumulation in the brain before administering the disease-modifying drug. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jack CR, Jr., Bennett DA, Blennow K, Carrillo MC, Dunn B, Haeberlein SB, et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease. Alzheimers Dement. 2018;14(4):535-62. Summary of the Invention [Problem to be solved by the invention]

[0005] However, while measuring the Aβ42 / 40 ratio can identify amyloid-β accumulation in the brain, it does not provide a means of determining the progression of AD. Furthermore, many patients (especially the very elderly) exhibit no cognitive impairment despite amyloid-β accumulation in the brain. Recent findings have demonstrated that phosphorylated tau (pTau181, 217, and 231) is an excellent biomarker for accurately predicting AD pathology at both symptomatic and asymptomatic stages and distinguishing AD from other neurodegenerative diseases. However, there is a need for a more accurate detection method for AD. Furthermore, in order to treat AD patients with anti-Aβ antibodies, there is a need for a diagnostic test for AD with high diagnostic accuracy that can distinguish between patients with mild cognitive impairment (MCI) due to AD and dementia due to AD from patients with other neurodegenerative diseases.

[0006] Therefore, an object of the present disclosure is to provide a new method that can be used to test for Alzheimer's disease and a test kit that can be used for said method. [Means for solving the problem]

[0007] In order to achieve the above object, the Alzheimer's disease testing method of the present disclosure (hereinafter also referred to as the "testing method") includes a measurement step of measuring the amount of target phosphorylated sites in two or more types of peptide fragments derived from phosphorylated tau protein in a subject sample using two or more measurement systems; a comparison step of comparing the magnitudes of the measured values ​​of the target phosphorylation portion, The measuring step a first measurement step of measuring the amount of the target phosphorylated site using a first measurement system comprising a first binding molecule that binds to the target phosphorylated site and a second binding molecule that binds to the non-phosphorylated site of the peptide fragment; a second measurement step of measuring the amount of the target phosphorylated site using a second measurement system comprising a third binding molecule that binds to the target phosphorylated site and a fourth binding molecule that binds to the non-phosphorylated site of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; The non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is shared by the two or more types of peptide fragments.

[0008] The Alzheimer's disease test kit (hereinafter also referred to as "test kit") of the present disclosure includes two or more measurement reagents for use in measuring the amount of target phosphorylated sites in two or more types of peptide fragments derived from phosphorylated tau protein in a subject sample, The first measurement reagent is a first binding molecule that binds to the target phosphorylation site; a second binding molecule that binds to the non-phosphorylated portion of the peptide fragment; The second measurement reagent is a third binding molecule that binds to the target phosphorylation site; a fourth binding molecule that binds to the non-phosphorylated portion of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; The non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is shared by the two or more types of peptide fragments. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide a new method that can be used to test for Alzheimer's disease and a test kit that can be used for said method. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing the common and non-consensus regions in peptide fragments of phosphorylated tau, and the binding sites of the first to fourth binding molecules that bind to the phosphorylated tau. [Figure 2] FIG. 2 is a graph showing the results of the ROC analysis in Example 1. [Figure 3]FIG. 3 is a graph (box plot) showing the results of the Aβ42 / 40 ratio and pTau ratio in AD and non-AD patients in Example 1. [Figure 4] FIG. 4 is a graph (box plot) showing the results of the Aβ42 / 40 ratio and the pTau ratio when AD and non-AD in Example 1 are further subdivided. [Figure 5] FIG. 5 is a graph showing the results of the ROC analysis in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Definition> As used herein, "tau protein" (hereinafter also referred to as "tau") refers to a protein that is expressed primarily in neurons in the central nervous system and contributes to the stabilization of microtubules. Human tau includes six isoforms: 3R0N (352 amino acids), 3R1N (381 amino acids), 3R2N (410 amino acids), 4R0N (383 amino acids), 4R1N (412 amino acids), and 4R2N (441 amino acids). Hereinafter, unless otherwise specified, the amino acid position in tau refers to the amino acid number corresponding to 4R2N (SEQ ID NO: 1). An example of the amino acid sequence of human tau (4R2N) is a protein consisting of the following amino acid sequence under GenBank accession number NP_005901.2.

[0012] Tau protein (SEQ ID NO: 1, NP_005901.2) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAKTPPPSGEPPKSGDRSGYSSPGTPGSRSRTPSLPTPPTTREPKKVAVVRTPPKSPSSAKSRLQTAPVMPDPLDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNHIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSTGSIDMVDSPQLATLADEVSASLAKQGL

[0013] As used herein, "phosphorylated tau protein" (hereinafter also referred to as "phosphorylated tau") refers to tau in which at least one amino acid residue suggested to be associated with Alzheimer's disease is phosphorylated. Examples of phosphorylated amino acid residues associated with Alzheimer's disease include threonine residues at positions 69, 71, 123, 153, 175, 181, 205, 212, 217, 231, 403, 414, and / or 427 in the tau, and threonine residues at positions 46, 68, 113, 184, 185, 191, 198, 199, 200, 201, 202, 203, 204, 205, 212, 217, 231, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 220, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 2 Serine residues at positions 202, 208, 210, 214, 235, 237, 238, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416, 422, 433, and / or 435, and / or tyrosine residues at positions 18, 197, and 394. The phosphorylated amino acid residues associated with Alzheimer's disease are, for example, the threonine residues at positions 175, 181, 205, 212, 217, and / or 231 in the tau protein, and / or the serine residues at positions 184, 185, 191, 198, 199, 202, 208, 210, 214, 235, 237, 238, 258, 262, 356, 396, 400, 404, 409, 412, 413, 416, and / or 422 in the tau protein, and / or the tyrosine residue at position 197. The phosphorylated amino acid residues associated with Alzheimer's disease are, for example, the threonine residues at positions 175, 181, 205, 212, 217, and / or 231, and / or the serine residues at positions 184, 185, 191, 198, 199, 202, 208, 214, 235, 262, 356, 396, 404, 409, 416, and / or 422 in the tau.The phosphorylated amino acid residue associated with Alzheimer's disease is preferably the threonine residue at positions 181, 212, 217, and / or 231, more preferably the threonine residue at positions 181 and / or 217, and even more preferably the threonine residue at position 181, because these residues are more highly associated with Alzheimer's disease. The number of phosphorylated amino acid residues in the phosphorylated tau is one or more. The phosphorylation of tau can be detected, for example, using a binding molecule that binds to the target phosphorylated amino acid residue.

[0014] As used herein, "amyloid β protein" (hereinafter also referred to as "Aβ") refers to a hydrophobic peptide protein consisting of 40 to 43 residues that is generated through two cleavage steps from the 695-amino acid (neuron-specific) amyloid precursor protein (APP). An example of the amino acid sequence of human APP is a protein consisting of the amino acid sequence encoded by the nucleic acid sequence of GenBank accession number NM_000484.4. It is known that Aβ is generated in vivo by degradation of APP by β-secretase and γ-secretase. Representative examples of Aβ peptide fragments include Aβ1-40 (Aβ40) consisting of 40 amino acid residues and Aβ1-42 (Aβ42) consisting of 42 amino acid residues. Aβ42 is highly insoluble and aggregative, and is thought to accumulate in brain tissues, contributing to Alzheimer's disease.

[0015] As used herein, "Alzheimer's disease" (hereinafter also referred to as "AD") refers to a neurodegenerative disease characterized by extracellular amyloid plaques (senile plaques) in the brain and neurofibrillary tangles (AD-related pathological changes) associated with abnormal phosphorylation of intracellular tau protein. AD is the most common cause of dementia, often develops in old age, and its main symptom is slowly progressive cognitive impairment, primarily memory loss. As used herein, AD may refer to either AD dementia (dementia caused by Alzheimer's disease) or MCI due to AD (mild cognitive impairment due to Alzheimer's disease). As used herein, "non-AD" includes patients with dementia or cognitive impairment other than Alzheimer's disease (dementia of the Alzheimer's type), as well as patients with neurological diseases with normal cognitive function.

[0016] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and particularly includes humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, hamsters, rabbits, goats, cows, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions. The subject may be, for example, an AD patient or a patient suspected of having AD. The suspected AD patient may be, for example, a subject who is subjectively suspected by the subject himself or herself, or may be a subject who has been diagnosed by a doctor or other medical professional as being suspected of having AD or who may be at risk of having AD based on symptoms or clinical findings. Examples of subjects who are subjectively suspected include those who have some subjective symptoms or who wish to undergo a preventive medical examination. Examples of clinical findings include findings based on cognitive function tests, brain MRI images, etc.

[0017] As used herein, a "sample" may be one that contains phosphorylated tau or one that may potentially contain phosphorylated tau. Examples of the sample include biological samples such as biological samples or specimens. Examples of the biological sample include samples containing body fluids, cells, tissues, organs, etc., and specific examples include cerebrospinal fluid, feces, whole blood, serum, plasma, spinal fluid, aspirate, bile, etc. The biological sample is preferably cerebrospinal fluid; or a blood sample such as whole blood, serum, or plasma. The sample may be liquid or solid. When the sample is solid, in the present disclosure, it is preferable to prepare a liquid sample by mixing the solid sample with a liquid. Examples of the liquid include water; physiological saline; and buffers such as Hank's buffer solution, Good's buffer solution (HEPES buffer, Tricine buffer, etc.), Tris buffer, phosphate buffer, and glycine buffer.

[0018] As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., test reagents, diagnostic reagents, test reagents, labels, substrates, instructions, etc.) are provided separately in two or more compartments. The kit can be suitably used to provide a composition that is not provided in a mixed state, but is preferably mixed immediately before use, for reasons of stability, etc. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., test reagents, diagnostic reagents, test reagents, etc.), or instructions or instructions describing the processing of the components. As used herein, when the kit is used as a reagent kit, the kit may also include instructions, etc., describing how to use the test reagent, diagnostic reagent, etc.

[0019] As used herein, "instructions" or "instructions" refer to written instructions to a physician or other user on how to use the present disclosure. The instructions, for example, describe instructions on how to use the testing method or kit of the present disclosure. The instructions may be prepared in accordance with a format specified by a regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, the European Medicines Agency (EMA) in Europe, etc.), and may clearly state that they have been approved by the regulatory agency. The instructions may be a so-called package insert, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, email).

[0020] As used herein, the terms "protein," "peptide," or "polypeptide" refer to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The polypeptide is, for example, a peptide having a length of 10 amino acids or more.

[0021] As used herein, "nucleic acid," "polynucleotide," or "oligonucleotide" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule. The polynucleotide may be composed of naturally occurring nucleotides, modified or artificial nucleotides, or both.

[0022] As used herein, the term "label" refers to a label used to distinguish a molecule or substance of interest from other molecules or substances. Examples of the label include fluorescent labels such as fluorescent dyes or fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine), chemiluminescent labels such as luciferin and aequorin, luminescent substances such as luminol and acridinium derivatives, electroluminescent substances such as ruthenium complexes, enzyme labels such as horseradish peroxidase, alkaline phosphatase, β-galactosidase (β-gal), glucose oxidase, and luciferase, and the like. 3 H, 14 C. 32 P, 35 S, 125 Radioisotope (RI) labels such as I;

[0023] As used herein, a "binding molecule" refers to a molecule capable of binding to a predetermined molecule. Examples of the binding molecule include nucleic acid molecules, proteins, and sugar chains capable of binding to the predetermined molecule. Specific examples of the binding molecule include aptamers, antibodies, receptors, and ligands capable of binding to the predetermined molecule. The binding molecule may be, for example, a known binding molecule capable of binding to the predetermined molecule, or a newly prepared binding molecule prepared by SELEX, phage display, or the like. The "antibody" refers to a protein containing one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Examples of the antibody include polyclonal antibodies and monoclonal antibodies. Examples of the antibody isotype include IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), IgM, IgA (e.g., IgA1, IgA2, etc.), IgE, IgD, IgY, etc. The antibody may be derived from animals such as mammals (e.g., mouse, rat, hamster, rabbit, goat, cow, horse, camel, and alpaca); birds (e.g., chicken and ostrich); and cartilaginous fish (e.g., shark). The antibody may be, for example, a camelid-derived heavy chain antibody (VHH antibody), a cartilaginous fish-derived immunoglobulin new antigen receptor (IgNAR), an antibody fragment (e.g., Fab, Fab', F(ab')2, single-domain antibody (nanobody), etc.), or a recombinant antibody (e.g., scFv, disulfide-linked Fv (dsFv), diabody, minibody, etc.). The antibody may be an antibody-like molecule (e.g., affibody, anticalin, DARPins, monobody, etc.) produced by molecular biology techniques (e.g., phage display) and / or by protein engineering techniques using existing protein motifs.

[0024] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequences using appropriate sequence conversion software, etc.

[0025] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0026] <Alzheimer's disease test kit> In one embodiment, the present disclosure provides a kit that can be used to test for Alzheimer's disease. The Alzheimer's disease test kit of the present disclosure includes two or more measurement reagents used to measure the amount of a target phosphorylated site in two or more peptide fragments derived from phosphorylated tau protein in a subject sample, wherein the first measurement reagent includes a first binding molecule that binds to the target phosphorylated site and a second binding molecule that binds to an unphosphorylated site in the peptide fragment, the second measurement reagent includes a third binding molecule that binds to the target phosphorylated site and a fourth binding molecule that binds to the unphosphorylated site in the peptide fragment, the unphosphorylated site to which the second binding molecule binds is a peptide region that is absent in at least one of the two or more peptide fragments, and the unphosphorylated site to which the fourth binding molecule binds is a peptide region that is common to the two or more peptide fragments.

[0027] As a result of extensive research, the present inventors have come up with the idea that the degree of fragmentation of peptide fragments derived from phosphorylated tau may vary depending on the onset or progression of Alzheimer's disease. Specifically, the present inventors have come up with the idea that the onset or progression of AD may cause the fragmentation of the phosphorylated protein to progress, resulting in the generation of peptide fragments of the phosphorylated protein with smaller molecular weights, as shown in Figure 1, and the existence of peptide fragments with different terminal positions. As a result of further research, the present inventors have found that a magnitude correlation exists between a measurement (first measurement) using a binding molecule that binds to a phosphorylated site in a peptide fragment of phosphorylated tau and a binding molecule that binds to a peptide region (non-common region) that is not present in at least one type of peptide fragment, as shown in Figure 1, and a measurement (second measurement) using a binding molecule that binds to a phosphorylated site in a peptide fragment of phosphorylated tau and a binding molecule that binds to a peptide region (common region) that is common to two or more types of peptide fragments; specifically, the second measurement is greater than the first measurement. They then discovered that AD can be tested by using the magnitude relationship (e.g., ratio or difference) between the first measurement value and the second measurement value as an index, and thus established the present disclosure. Furthermore, as described below, when AD patients and non-AD patients are tested using the ratio between the first measurement value and the second measurement value, the ratio between the first measurement value and the second measurement value functions as an index with diagnostic accuracy equal to or higher than that of Aβ42 / 40, a commonly used AD marker. Therefore, the test method of the present disclosure and the test kit for use in the test method can be suitably used to test for AD. Furthermore, since the test method of the present disclosure can distinguish between AD patients and non-AD patients, it can also be referred to as, for example, a method for distinguishing AD.

[0028] The first measurement reagent contains the first binding molecule and the second binding molecule. In the first measurement reagent, the first binding molecule and the second binding molecule may be contained in a mixed state or may be contained separately. In the first measurement reagent, the first binding molecule and the second binding molecule may each be composed of one or more types.

[0029] The first binding molecule binds to the target phosphorylation site. The target phosphorylation site refers to a phosphorylated amino acid residue in the phosphorylated tau that is to be detected. The first binding molecule may, for example, recognize and bind only to the phosphorylated amino acid residue in the tau that is to be detected, or may recognize and bind to a peptide region containing the phosphorylated amino acid residue, i.e., the phosphorylated amino acid residue and its surrounding peptide region. The first binding molecule preferably binds to a peptide containing the phosphorylated amino acid residue that is to be detected, but does not bind to a peptide from which the phosphate group in the phosphorylated amino acid residue that is to be detected has been eliminated. Examples of the target phosphorylation site include a phosphorylated serine residue, a phosphorylated threonine residue, and / or a phosphorylated tyrosine residue. The target phosphorylation site is, for example, a phosphorylated serine residue in the phosphorylated tau, and the serine residue is located at positions 46, 68, 113, 184, 185, 191, 198, 199, 202, 208, 210, 214, 235, 237, 238, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416, 422, 433, and 435 in the tau. or serine residues selected from the group consisting of positions 184, 185, 191, 198, 199, 202, 208, 214, 235, 262, 356, 396, 404, 409, 416, and 422 in the tau, preferably serine residues selected from the group consisting of positions 198, 199, 202, 235, 400, 404, 412, 413, and 416.The target phosphorylation site is, for example, a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is a threonine residue selected from the group consisting of positions 69, 71, 123, 153, 175, 181, 205, 212, 217, 231, 403, 414, and 427 in the tau protein, or a threonine residue selected from the group consisting of positions 69, 175, 181, 205, 212, 217, and 231 in the tau protein, preferably a threonine residue selected from the group consisting of positions 181, 212, 217, and 231. The target phosphorylation site is, for example, a phosphorylated tyrosine residue in the phosphorylated tau protein, and the tyrosine residue is a tyrosine residue selected from the group consisting of positions 18, 197, and 394 in the tau. The target phosphorylation site is preferably a phosphorylated threonine residue in the phosphorylated tau protein, for example, because of its higher association with Alzheimer's disease, and the threonine residue is preferably the threonine residue at positions 181, 212, 217, and / or 231 in the tau, more preferably the threonine residue at positions 181 and / or 217, and even more preferably the threonine residue at position 181. The first binding molecule binds, for example, to one or more target phosphorylation sites in the phosphorylated tau, and preferably binds to one target phosphorylation site.

[0030] The first binding molecule is preferably a binding molecule specific to the target phosphorylation site. The first binding molecule is preferably an antibody or an antigen-binding fragment thereof specific to the target phosphorylation site. When the first binding molecule is, for example, a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495, 1975). Furthermore, antibodies specific to the target phosphorylation site are commercially available, and commercially available antibodies can also be used. Specific examples of antibodies that bind to the phosphorylated threonine residue (position 175) include polyclonal antibodies (MM-0147-P, manufactured by MEDIMABS). Examples of antibodies that bind to the phosphorylated threonine residue (position 181) include EPR23506-107 (Abcam), D9F4G (Cell signaling), AT270 (Furjibio Europe), 1E7 (Merk), and ADx252 (ADx NeuroSciences). Examples of antibodies that bind to the phosphorylated serine residue (position 199) include polyclonal antibody (44-734G, Thermo Fisher Scientific), polyclonal antibody (#29957, Cell signaling), polyclonal antibody (ab4749, Abcam), polyclonal antibody (AB9652, Merck), and 5B8-1E2 (Fujifilm Wako Pure Chemical Industries, Ltd.). Examples of antibodies that bind to the phosphorylated serine or threonine residue (position 202 (S) or 205 (T)) include AT8 (manufactured by Fujirebio Europe). Examples of antibodies that bind to the phosphorylated threonine residue (position 212 or 214) include AT100 (manufactured by Thermo Fisher Scientific).Examples of antibodies that bind to the phosphorylated threonine residue (position 217) include EPR24654-24 (Abcam), E9Y4S (Cell signaling), IBA493 and IBA413 (Eli Lilly), PT3 (Janssen), and polyclonal antibody (44-744, Thermo Fisher Scientific). Examples of antibodies that bind to the phosphorylated threonine residue (position 231) include EPR2488 (Abcam), 44-746G (Thermo Fisher Scientific), PHF-6 (Merk), AT180 (Fujirebio Europe), ADx253 (ADx NeuroSciences), polyclonal antibody (#71429, Cell signaling), and polyclonal antibody (AB9668, Merck). Examples of antibodies that bind to the phosphorylated serine residue (position 396) include PHF13.6 (manufactured by Thermo Fisher Scientific) and 5HCLC (manufactured by Thermo Fisher Scientific). Examples of antibodies that bind to the phosphorylated serine residue (position 396 or 404) include PFH1 (manufactured by Creative Biolabs). Examples of antibodies that bind to the phosphorylated serine residue (position 413) include the antibodies described in WO 2021 / 262791. Examples of antibodies that bind to the phosphorylated serine residue (position 416) include the antibodies described in WO 2013 / 180238.

[0031] The second binding molecule binds to the non-phosphorylated portion of the peptide fragment. The non-phosphorylated portion to which the second binding molecule binds is a peptide region (non-common region) that is not present in at least one of the two or more peptide fragments. Specifically, the non-phosphorylated portion to which the second binding molecule binds can be determined, for example, by aligning peptide fragments of phosphorylated tau containing the target phosphorylation portion, as shown in FIG. 1, and setting the non-phosphorylated portion to a region that is not present in at least one of the aligned peptide fragments. The non-common region may be determined, for example, from the amino acid sequence of a known peptide fragment of phosphorylated tau. Alternatively, the non-common region may be determined, for example, by measuring peptide fragments in a blood sample or cerebrospinal fluid of the AD patient. In this case, for example, a peptide fragment of phosphorylated tau containing the target phosphorylation portion is extracted from a blood sample or cerebrospinal fluid of the AD patient using a first binding molecule that binds to the target phosphorylation portion or a region surrounding the target phosphorylation portion. Next, the amino acid sequences of the extracted peptide fragments are identified using a known amino acid sequence identification method such as liquid chromatography tandem mass spectrometry (LC / MS / MS), and the peptide fragments are aligned based on the obtained amino acid sequences. The alignment can be performed by identifying a region that is absent from at least one of the aligned peptide fragments. Specifically, when the target phosphorylation site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at positions 181 and / or 217, the non-phosphorylated site to which the second binding molecule binds is, for example, the peptide region from positions 1 to 180 in the tau protein, preferably positions 1 to 167, 111 to 180, 124 to 180, 111 to 167, or 124 to 167, and particularly preferably positions 159 to 163. The second binding molecule is a binding molecule that binds to part or all of the peptide region, and is configured, for example, so as not to compete with the first binding molecule for binding to the peptide fragment.

[0032] The second binding molecule is, for example, a peptide region that is absent in at least one peptide fragment and present in at least one peptide among the two or more peptide fragments. The second binding molecule is preferably a peptide region that is absent in two or more, three or more, four or more, or five or more peptide fragments among the two or more peptide fragments, and optionally a peptide region that is present in at least one peptide, because this can broaden the dynamic range of the first measurement value.

[0033] The second binding molecule is preferably a binding molecule specific to a non-common region of the peptide fragments, i.e., a peptide region that is not present in at least one of the two or more peptide fragments. The second binding molecule is preferably an antibody or an antigen-binding fragment thereof that is specific to the non-common region of the peptide fragments, i.e., a peptide region that is not present in at least one of the two or more peptide fragments. When the antibody against the non-common region is, for example, a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495, 1975). Furthermore, antibodies against the non-common region are commercially available, and commercially available antibodies can also be used. Specific examples of the second binding molecule include LRL (Eli Lilly and Company, epitope: positions 111-130), 4G10-E2 (Eli Lilly and Company, epitope: positions 111-130), ADx204 (ADx NeuroSciences, epitope: positions 6-18), Tau12 (Quanterix, epitope: positions 6-18), HT7 (Thermo Fisher Scientific, epitope: positions 159-163), and HT43 (Janssen, epitope: positions 7-20), when the target phosphorylation site is a phosphorylated threonine residue in the phosphorylated tau protein and the threonine residue is the threonine residue at position 181.

[0034] The first measurement reagent may contain, for example, another binding molecule that binds to a region other than the phosphorylated peptide region to which the first binding molecule binds and the peptide region to which the second binding molecule binds. The other binding molecule binds, for example, to a non-phosphorylated portion of the peptide fragment. The other binding molecule contained in the first measurement reagent may, for example, be a binding molecule that binds to the non-common region. When the other binding molecule binds to the non-common region, it is preferable that the other binding molecule binds to a peptide region other than the peptide region to which the second binding molecule binds.

[0035] The first measurement reagent may be, for example, a commercially available kit. When the target phosphorylated site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at position 181, examples of the kit include INNOTEST (registered trademark) Phospho-Tau (181P) (manufactured by Fujirebio Inc.) and S-PLEX Human Tau (pT181) Kit (manufactured by MSD).

[0036] The second measurement reagent includes the third binding molecule and the fourth binding molecule. In the second measurement reagent, the third binding molecule and the fourth binding molecule may be contained in a mixed state or may be contained separately. In the second measurement reagent, the third binding molecule and the fourth binding molecule may each be composed of one or more types, for example.

[0037] The third binding molecule that binds to the target phosphorylation site may be, for example, the same binding molecule as the first binding molecule, or a different binding molecule. The explanation of the third binding molecule can be incorporated by replacing "first binding molecule" with "third binding molecule" in the explanation of the first binding molecule. The first binding molecule and the third binding molecule may, for example, bind to the same target phosphorylation site or different target phosphorylation sites. That is, the first binding molecule and the third binding molecule may, for example, bind to the same phosphorylated amino acid residue or different target phosphorylation sites, with the former being preferred.

[0038] The fourth binding molecule binds to the non-phosphorylated portion of the peptide fragment. The non-phosphorylated portion to which the fourth binding molecule binds is a peptide region (common region) shared by the two or more peptide fragments. Specifically, the non-phosphorylated portion to which the second binding molecule binds can be set to a region shared by each of the aligned peptide fragments by aligning peptide fragments of phosphorylated tau containing the target phosphorylated portion, as shown in FIG. 1 . The common region may be set, for example, from the amino acid sequence of a known peptide fragment of phosphorylated tau. Alternatively, the common region may be set, for example, by measuring peptide fragments in a blood sample or cerebrospinal fluid of the AD patient. In this case, for example, a third binding molecule that binds to the target phosphorylated portion is used to extract a peptide fragment of phosphorylated tau containing the target phosphorylated portion from a blood sample or cerebrospinal fluid of the AD patient. Next, in the setting, for example, the amino acid sequences of the extracted peptide fragments are identified using a known amino acid sequence identification method such as the LC / MS / MS, and the peptide fragments are aligned based on the obtained amino acid sequences. Then, in the setting, a region that is common to the two or more types of peptide fragments is identified in each of the aligned peptide fragments. Specifically, when the target phosphorylated site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at position 181, the non-phosphorylated site is, for example, a peptide region from positions 182 to 441 in the tau protein, preferably a peptide region from positions 182 to 224, 182 to 223, 182 to 220, 182 to 210, 185 to 441, 185 to 224, 185 to 223, 185 to 220, or 185 to 210, and particularly preferably a peptide region from positions 194 to 198.When the target phosphorylated site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at position 217, the non-phosphorylated site is, for example, a peptide region from positions 182 to 441 in the tau protein, preferably a peptide region from positions 182 to 224, 182 to 223, 182 to 220, 182 to 210, or 182 to 209, and particularly preferably a peptide region from positions 194 to 198. The fourth binding molecule is a binding molecule that binds to a part or all of the peptide region. The fourth binding molecule is preferably configured so as not to compete with the third binding molecule for binding to the peptide fragment.

[0039] The fourth binding molecule is preferably a binding molecule specific to a common region of the peptide fragments, i.e., a peptide region shared by the two or more peptide fragments. The fourth binding molecule is preferably an antibody or an antigen-binding fragment thereof specific to the common region of the peptide fragments, i.e., a peptide region shared by the two or more peptide fragments. When the antibody specific to the common region is, for example, a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495, 1975). Furthermore, antibodies specific to the common region are commercially available, and commercially available antibodies can also be used. Specifically, when the target phosphorylation site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at position 181, the fourth binding molecule can be, for example, BT2 (Invitrogen, epitope: positions 194-198).

[0040] The second measurement reagent may contain, for example, another binding molecule that binds to a region other than the phosphorylated peptide region to which the third binding molecule binds and the peptide region to which the fourth binding molecule binds. The other binding molecule binds, for example, to a non-phosphorylated portion of the peptide fragment. The other binding molecule contained in the second measurement reagent may be, for example, a binding molecule that binds to the non-common region or the common region. When the other binding molecule binds to the non-common region, the other binding molecule is preferably the second binding molecule. When the other binding molecule binds to the common region, the other binding molecule is preferably a binding molecule different from the fourth binding molecule and / or a binding molecule that binds to a peptide region other than the peptide region to which the fourth binding molecule binds.

[0041] The second measurement reagent may be, for example, a commercially available kit. When the target phosphorylated site is a phosphorylated threonine residue in the phosphorylated tau protein, and the threonine residue is the threonine residue at position 181, the kit may be, for example, Lumipulse (registered trademark) Phosphorylated Tau 181 (manufactured by Fujirebio Inc.).

[0042] When one of the first and second measurement reagents contains another binding molecule, it is preferable that the other measurement reagent does not contain another binding molecule. Specifically, when the second measurement reagent contains the second binding molecule as the other binding molecule, it is preferable that the first measurement reagent does not contain the other binding molecule. Furthermore, it is preferable that the first measurement reagent and the second measurement reagent contain at least one different binding molecule.

[0043] Any one or more of the first to fourth binding molecules, i.e., one, two, three, or four, may be supported on a carrier. That is, the test kit of the present disclosure may include, for example, a carrier supporting any one or more of the first to fourth binding molecules. The carrier may be, for example, particles such as magnetic particles or beads; membranes such as nitrocellulose membranes; substrates such as glass, plastic, or metal; plates such as multiwell plates; tubes, test tubes, capillaries, nanopillars, or microchannels; and is preferably a particle due to its excellent operability. Each binding molecule may be provided in a form impregnated in a medium such as filter paper. Each binding molecule may be configured to be supported on a carrier during measurement of phosphorylated tau peptide fragments. That is, in the test kit of the present disclosure, each binding molecule may exist in a separate state and coexist during measurement, thereby supporting each binding molecule on the carrier. In this case, the test kit of the present disclosure may, for example, bind a first component of a pair of affinity substances to each of the binding molecules, bind a second component of the pair of affinity substances to the carrier, and support each of the binding molecules on the carrier via affinity binding between the first and second components. The pair of affinity substances includes the first and second components, and the first and second components are a combination of substances that exhibit specific binding ability to each other, and can also be referred to as, for example, a tag and a binding partner.

[0044] The method for immobilizing each binding molecule on the carrier can be a conventionally known method or a method based thereon, depending on the type of binding molecule. The binding molecules may be immobilized directly or indirectly on the surface of the carrier. Examples of direct immobilization include a method in which an active group is added to the carrier, and the resulting carrier, or a carrier having an active group, is used, and each binding molecule is bound to the carrier via a covalent bond formed by reaction between the active group and each binding molecule. Examples of indirect immobilization include a method using a pair of affinity substances. Examples of the pair of affinity substances include a combination of biotin and avidin or streptavidin, a combination of nickel and a His tag, or a combination of an epitope tag, such as a flag™ tag, HA tag, T7 tag, V5 peptide tag, and / or Myc tag, and an antibody against the tag. Furthermore, each binding molecule may be supported on the carrier by ionic bonding with the carrier or by adsorption to the carrier. When each of the binding molecules is indirectly supported on a carrier, for example, each of the binding molecules and the carrier may be contained in the same container or in separate containers.

[0045] Any one or more of the first to fourth binding molecules, i.e., one, two, three, or four, may have a label. In this case, the target phosphorylated site can be detected via the first to fourth binding molecules by detecting the label. In the first measurement reagent, at least one of the first binding molecule and the second binding molecule preferably has a label, and the other has a carrier. Furthermore, in the second measurement reagent, at least one of the third binding molecule and the fourth binding molecule preferably has a label, and the other has a carrier. The method for introducing a label into each binding molecule can be carried out by appropriately adopting a conventionally known method or a method equivalent thereto, depending on the type of each binding molecule. The label may be directly or indirectly bound. Examples of the method for binding the label include the same methods as those exemplified as the method for immobilizing each binding molecule on the carrier.

[0046] When any one or more of the first to fourth binding molecules has a label, the test kit of the present disclosure may have a substrate capable of reacting with the label. In this case, the label is, for example, the enzyme label described above, and the substrate is a substance capable of reacting with the enzyme label. The substrate may be solid or liquid. When the substrate is liquid, the substrate may also be referred to as a substrate liquid.

[0047] The binding molecules in the first and second test reagents of the test kit of the present disclosure may be in the form of a solution (liquid or gel) dissolved or dispersed in a buffer solution or the like, or may be in a solid form such as powder or granules, obtained by freeze-drying the liquid dissolved in the buffer solution or the like. Examples of the buffer solution include Tris buffers such as Tris-HCl buffer, Tris-EDTA (TE) buffer, TAE buffer, TBE buffer, and Tris-buffered saline; phosphate buffers such as phosphate-buffered saline; carbonate buffers such as carbonate-sodium bicarbonate buffer; and Good's buffers such as MES, ADA, PIPES, TAPS, CAPS, ACES, cholamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycineamide, and bicine. The pH of the solution is, for example, 4 to 9.5, preferably 5 to 9 or 5.5 to 8.5, and more preferably 6 to 8. The pH can be adjusted using, for example, the buffer solution, an acidic substance such as hydrochloric acid, or an alkaline substance such as sodium hydroxide. The solution may further contain a water-soluble polymer such as bovine serum albumin (BSA), a chelating agent such as EDTA, a sugar such as sucrose, or a preservative such as sodium azide.

[0048] The test kit of the present disclosure may further include a preparation of a phosphorylated tau peptide fragment. The preparation of a phosphorylated tau peptide fragment is an aqueous solution containing one or more predetermined concentrations of phosphorylated tau peptide fragments, or a powder of phosphorylated tau peptide fragments (e.g., a lyophilized product). The preparation of a phosphorylated tau peptide fragment is useful, for example, as a control. Furthermore, by using the preparation of a phosphorylated tau peptide fragment, for example, in the method for measuring phosphorylated tau peptide fragments described below, a calibration curve corresponding to the concentration of phosphorylated tau peptide fragments can be prepared, and the concentration of phosphorylated tau peptide fragments in a sample can be analyzed. The phosphorylated tau peptide fragments and their phosphorylated sites in the preparation can be set, for example, according to the binding regions of the first to fourth binding molecules.

[0049] The test kit of the present disclosure may include, for example, a diluent for diluting a sample (specimen diluent), a diluent for diluting a first binding molecule (first binding molecule diluent), a diluent for diluting a second binding molecule (second binding molecule diluent), a diluent for diluting a third binding molecule (third binding molecule diluent), a diluent for diluting a fourth binding molecule (fourth binding molecule diluent), a washing solution for washing the first complex or the second complex formed by the reaction between the sample and the first measurement reagent or the second measurement reagent, a pretreatment solution (treatment solution), etc.

[0050] The diluent contains, for example, the buffer solution, and the washing solution contains, for example, the buffer solution and a nonionic surfactant.

[0051] In the test kit of the present disclosure, each reagent or component may be in a solid form such as powder or granules, or in a liquid form such as a slurry (suspension), jelly, or solution.

[0052] In the test kit of the present disclosure, for example, each component may be contained separately, or some or all of the components may be contained in a mixed or unmixed state. In the test kit of the present disclosure, when all reagents are contained in a single container in a mixed or unmixed state, the test kit of the present disclosure can also be referred to as, for example, a test reagent for Alzheimer's disease.

[0053] The test kit of the present disclosure may further include, for example, containers for storing the components of the kit. In this case, the test kit of the present disclosure may be provided with each component contained in a different container (e.g., a tube, a plate, etc.). The test kit of the present disclosure may also be provided in the form of a device. In this case, some or all of the components may be provided in a form contained in the device. When some of the components are provided in a form contained in the device, the remaining components of the kit may be provided in a form not contained in the device, for example, in a form contained in a different container. In this case, the components not contained in the device may be used by being injected into the device when measuring the target phosphorylation site. Examples of the device structure include: 1) a device having a first area for mixing a sample with either the binding molecule in the first measurement reagent or the second measurement reagent to prepare a mixed solution, and a second area for contacting the prepared mixed solution with the other binding molecule in the first measurement reagent or the second measurement reagent to detect the target phosphorylated site; 2) a device having an area for mixing a sample with either the first measurement reagent or the second measurement reagent to detect the target phosphorylated site; and 3) a device having a flow path that allows mixing of the sample with the components (e.g., reaction solution, diluent, etc.) and an area for detecting the target phosphorylated site.

[0054] Test kits of the present disclosure may include, for example, instructions or instructions.

[0055] The test kit of the present disclosure includes, for example, the first measurement reagent and the second measurement reagent, and can be suitably used as a test kit, a test kit, or a research kit for measuring the first measurement value and the second measurement value. Furthermore, the test kit of the present disclosure can suitably carry out the AD testing method of the present disclosure described below.

[0056] <Alzheimer's disease testing methods> In another aspect, the present disclosure provides a method usable for testing for Alzheimer's disease. The method for testing for Alzheimer's disease of the present disclosure comprises a measurement step of measuring the amounts of target phosphorylated sites in two or more peptide fragments derived from phosphorylated tau protein in a subject sample using two or more measurement systems, and a comparison step of comparing the measured values ​​of the target phosphorylated sites, wherein the measurement step comprises a first measurement step of measuring the amounts of the target phosphorylated sites using a first measurement system comprising a first binding molecule that binds to the target phosphorylated site and a second binding molecule that binds to an unphosphorylated site of the peptide fragment, and a second measurement step of measuring the amounts of the target phosphorylated sites using a second measurement system comprising a third binding molecule that binds to the target phosphorylated site and a fourth binding molecule that binds to the unphosphorylated site of the peptide fragment, wherein the unphosphorylated site to which the second binding molecule binds is a peptide region that is absent in at least one of the two or more peptide fragments, and the unphosphorylated site to which the fourth binding molecule binds is a peptide region that is common to the two or more peptide fragments. The testing method of the present disclosure performs AD testing using the magnitude relationship between the first measurement value and the second measurement value, which functions as an indicator with diagnostic accuracy equal to or higher than that of Aβ42 / 40. Therefore, the testing method of the present disclosure can suitably test for AD. The testing method of the present disclosure can calculate a ratio that serves as an indicator of Alzheimer's disease. Therefore, the testing method of the present disclosure can also be said to be, for example, a method for providing an indicator of Alzheimer's disease.

[0057] In the measurement step, the amounts of target phosphorylated sites in two or more peptide fragments derived from phosphorylated tau protein are measured for a subject sample using two or more measurement systems. In the measurement step, for example, the subject sample is measured using the first measurement reagent, thereby measuring the measurement result using the first measurement system, i.e., the first measurement value (V1). In addition, in the measurement step, for example, the subject sample is measured using the second measurement reagent, thereby measuring the measurement result using the second measurement system, i.e., the second measurement value (V2). Therefore, in the measurement step, for example, a measurement system using the subject sample and the first measurement reagent and a measurement system using the subject sample and the second measurement reagent are prepared separately, and measurements are performed using each measurement system separately.

[0058] In the measurement step, the target phosphorylation site can be measured using the first measurement reagent and the second measurement reagent. In this case, the target phosphorylation site may be measured by an immunological technique. Examples of the immunological technique include sandwich ELISA, sandwich immunoassay, and immunochromatography. Furthermore, when the target phosphorylation site measurement reagent contains a label, examples of the immunological technique include fluorescent immunoassay (FIA), enzyme immunoassay (EIA), chemiluminescent immunoassay, chemiluminescent enzyme immunoassay, and radioimmunoassay (RIA), depending on the type of label.

[0059] In the measurement step, the first measurement step using the first measurement system can be carried out, for example, as follows. Specifically, the first measurement step involves, for example, contacting the target sample with the first binding molecule and the second binding molecule. As a result, the first measurement step forms a first complex between a peptide fragment containing the target phosphorylation site in the target sample and the first binding molecule and the second binding molecule (first complex formation step). In the first complex formation step, the order in which the sample is contacted with the first binding molecule and the second binding molecule is not particularly limited, as long as it is an order that allows the first complex to be formed. Specifically, in the first complex formation step, the sample may be contacted with the first binding molecule and the second binding molecule simultaneously, or the target sample may be contacted with the first binding molecule to form a complex, and then the complex may be contacted with the second binding molecule, or the target sample may be contacted with the second binding molecule to form a complex, and then the complex may be contacted with the first binding molecule. The contacting is preferably carried out in a liquid system containing water, physiological saline, the buffer solution, or the like.

[0060] In the first complex formation step, the first binding molecule or the second binding molecule may be directly immobilized on the carrier in advance, or may be supported on the carrier before, during, or after the formation of the first complex. In the latter case, the first binding molecule or the second binding molecule is indirectly immobilized on the carrier, for example. The indirect immobilization can be achieved, for example, by adding the above-mentioned pair of affinity substances to the first binding substance and the carrier.

[0061] In the first complex formation step, the contact conditions (e.g., temperature, time, pH) between the sample and the first binding molecule and / or the second binding molecule are not particularly limited as long as they allow the formation of the first complex. Specific examples of the contact temperature include 4 to 42°C, or 18 to 40°C. The contact time is, for example, 1 minute to 12 hours, 3 minutes to 120 minutes, or 5 to 60 minutes. The pH during the contact is, for example, 4 to 9.5, preferably 5 to 9 or 5.5 to 8.5, and more preferably 6 to 8.

[0062] In the first measurement step, for example, after a complex is formed between a peptide containing a target phosphorylation site in the sample and the first binding molecule, or after a complex is formed between a peptide containing a target phosphorylation site in the sample and the second binding molecule, it is preferable to separate these complexes from other components that do not form complexes, since this can improve the accuracy of measuring the target phosphorylation site. Optionally, it is more preferable to further wash the separated complex with the washing solution. Separation of the complex with the first binding molecule or the complex with the second binding molecule can be performed, for example, by solid-liquid separation. When the first binding molecule and / or the second binding molecule are supported on a carrier, separation of the first complex can be performed, for example, by separating a solid fraction containing the carrier from a liquid fraction. When the first binding molecule and / or the second binding molecule are supported on magnetic particles, separation of the complex with the first binding molecule or the complex with the second binding molecule can be performed by generating a magnetic field using a magnet or the like to separate a solid fraction containing the magnetic particles from a liquid fraction.

[0063] Furthermore, in the first measurement step, for example, after the first complex formation step, it is preferable to separate the first complex from other components that do not form a complex, since this can improve the measurement accuracy of the target phosphorylation site. Optionally, it is more preferable to further wash the separated first complex with the washing solution. Separation of the first complex can be carried out, for example, by solid-liquid separation. When the first binding molecule and / or the second binding molecule are supported on a carrier, separation of the first complex can be carried out, for example, by separating a solid fraction containing the carrier from a liquid fraction. When the first binding molecule and / or the second binding molecule are supported on magnetic particles, separation of the first complex can be carried out by generating a magnetic field using a magnet or the like to separate a solid fraction containing the magnetic particles from a liquid fraction.

[0064] The first complex measurement step measures the amount of the target phosphorylation site in the sample by detecting the first complex. Specifically, the first complex measurement step is, for example, a step of measuring the binding between a peptide fragment containing the target phosphorylation site in the sample, the first binding molecule, and the second binding molecule. Thus, in the first complex measurement step, by detecting the presence or absence of binding between the three, for example, the presence or absence of the target phosphorylation site in the sample can be analyzed (qualitatively determined), and by detecting the degree of binding between the three (amount of binding), for example, the amount of the target phosphorylation site in the sample can be analyzed (quantitatively determined).

[0065] The method for measuring the binding between the peptide fragment containing the target phosphorylation site, the first binding molecule, and the second binding molecule is not particularly limited. For example, any conventional method for measuring binding between substances can be used, such as SPR or fluorescence polarization. Furthermore, when the first binding molecule and / or the second binding molecule have a label, the first complex measurement step may measure the binding between the peptide fragment containing the target phosphorylation site, the first binding molecule, and the second binding molecule by directly or indirectly detecting the label in the first complex. The detection of the label can be determined appropriately depending on, for example, the type of label. Furthermore, when the first binding molecule and / or the second binding molecule have a label, it is preferable that at least one of the first binding molecule and the second binding molecule has a label, and the other is directly immobilized on the support or indirectly supported on the support. In this case, in the first complex measurement step, for example, the binding between the peptide fragment containing the target phosphorylated site, the first binding molecule, and the second binding molecule can be measured by directly or indirectly detecting the label in the first complex formed on the carrier.

[0066] In the first complex measurement step, for example, the first complex of the peptide fragment containing the target phosphorylation site, the first binding molecule, and the second binding molecule may be separated from other components that do not form a complex, the separated first complex may be washed with the washing solution, and then the label may be released from the first complex. The released label may be detected, thereby measuring the binding between the peptide fragment containing the target phosphorylation site, the first binding molecule, and the second binding molecule.

[0067] Next, in the measurement step, the second measurement step using the second measurement system can be carried out, for example, as follows. Specifically, the second measurement step involves, for example, contacting the target sample with the third binding molecule and the fourth binding molecule. As a result, the second measurement step forms a second complex between the peptide fragment containing the target phosphorylation site in the target sample and the third binding molecule and the fourth binding molecule (second complex formation step). The explanation of the second complex measurement step can be applied, for example, by replacing the "first binding molecule" with the "third binding molecule," the "second binding molecule" with the "fourth binding molecule," and the "first complex" with the "second complex" in the explanation of the first complex formation step.

[0068] The second complex measurement step is, for example, a step of measuring a second complex of a peptide fragment containing a target phosphorylated site, the third binding molecule, and the fourth binding molecule in the sample. Thus, in the second complex measurement step, the presence or absence of the target phosphorylated site in the sample can be analyzed (qualitatively) by detecting the presence or absence of binding between the two molecules, and the amount of the target phosphorylated site in the sample can be analyzed (quantitatively) by detecting the degree of binding between the two molecules (amount of binding). The explanation of the second complex measurement step can be applied, for example, by replacing "first binding molecule" with "third binding molecule," "second binding molecule" with "fourth binding molecule," and "first complex" with "second complex."

[0069] Next, in the comparison step, the measured values ​​of the target phosphorylation site are compared in magnitude. The comparison of the measured values ​​can be performed, for example, by calculating the ratio or difference between the measured value (V1) obtained in the first measurement step and the measured value (V2) obtained in the second measurement step. The ratio of the measured values ​​may be, for example, the ratio of V2 to (V1) (V2 / V1), or V1 / V2, or a ratio calculated by a method similar to or modified thereto. The difference of the measured values ​​may be, for example, the difference of V2 to (V1) (V2-V1), or V1-V2, or a difference calculated by a method similar to or modified thereto. The comparison of the measured values ​​may be calculated, for example, as (V2-V1) / V1.

[0070] In this way, the testing method of the present disclosure can determine the magnitude relationship that serves as an indicator of Alzheimer's disease.

[0071] Furthermore, in the testing method of the present disclosure, as described above, the second measurement system may include the second binding molecule in addition to the fourth binding molecule. In this case, in the testing method of the present disclosure, for example, in the second measurement step, the target sample is contacted with the third binding molecule, the fourth binding molecule, and the second binding molecule. As a result, in the second measurement step, in addition to the second complex, a third complex (or the first complex if the third binding molecule is the same as the first binding molecule) containing, for example, a peptide fragment containing the target phosphorylation site in the target sample, the third binding molecule, and the second binding molecule is also formed. Therefore, in the second complex measurement step, for example, in addition to the second complex, the third complex (or the first complex if the third binding molecule is the same as the first binding molecule) is also measured, and this is designated as V2. In this case, the comparison step may also involve comparing the magnitudes of the measured values ​​of the target phosphorylation site.

[0072] In the testing method of the present disclosure, the sample may be diluted with the specimen dilution solution prior to the measurement step. The volume of the dilution solution used to dilute the sample can be appropriately determined depending on the volume of the sample and the intended use of the sample (e.g., qualitative measurement, quantitative measurement, etc.).

[0073] The testing method of the present disclosure may include, for example, a testing step (evaluation step) of testing (evaluating) the possibility of the subject having Alzheimer's disease or the presence or absence of the disease based on the magnitude relationship between the measured values ​​of the target phosphorylation sites. The magnitude relationship may be, for example, the ratio of the measured values, the difference of the measured values, or a value calculated using a method similar to or modified thereto. Hereinafter, an example will be described in which the ratio of the measured values ​​is used as the magnitude relationship, but the testing method of the present disclosure is not limited thereto and can be similarly carried out using other magnitude relationships, such as the difference of the measured values. When the difference of the measured values ​​is used instead of the ratio of the measured values, the following description can be used by replacing "ratio of measured values" with "difference of measured values," "V2 / V1" with "V2-V1," and "V1 / V2" with "V1-V2," except for the specific numerical values ​​of the threshold values.

[0074] The evaluation can be performed, for example, by comparing the ratio of the measured values ​​of the target phosphorylation sites with a threshold. The threshold can be, for example, the ratio of the measured values ​​of the target phosphorylation sites obtained using a sample from a healthy individual; the ratio of the measured values ​​of the target phosphorylation sites obtained using a sample from a patient diagnosed with a neurological disease other than AD but with cognitive impairment and a patient with a neurological disease but normal cognitive function (non-AD patient); the ratio of the measured values ​​of the target phosphorylation sites obtained using a sample from an AD patient; or a threshold set based on these. The threshold can be set, for example, by performing ROC analysis using the ratio of the measured values ​​of the target phosphorylation sites obtained using a sample from the non-AD patient and the measured values ​​of the target phosphorylation sites obtained using a sample from the AD patient, and using the Youden Index or the like. Alternatively, the threshold can be set, for example, by performing ROC analysis using the ratio of the measured values ​​of the target phosphorylation sites obtained using a sample from the healthy individual and the measured values ​​of the target phosphorylation sites obtained using a sample from the AD patient, and using the Youden Index or the like. The threshold can also be set by, for example, determining the presence or absence of amyloid-β accumulation using a method for examining the presence or absence of amyloid accumulation in the brain, such as amyloid PET (Positron Emission Tomography), and performing ROC analysis using the ratio of measured values ​​of the target phosphorylated site obtained using a sample from a patient with amyloid-β accumulation (e.g., an amyloid PET-positive patient) and / or the ratio of measured values ​​of the target phosphorylated site obtained using a sample from a patient without amyloid-β accumulation (e.g., an amyloid PET-negative patient), and using the Youden Index or the like. Specifically, when a reference based on a doctor's diagnosis is used, the V2 / V1 threshold for distinguishing (differentiating) AD patients from non-AD patients can be set, for example, between 1.081 and 1.362 or between 1.025 and 1.362 (when the sensitivity and specificity are 0.8 or higher). Furthermore, when using amyloid PET results as a reference, the V2 / V1 threshold for distinguishing between AD and non-AD patients can be set, for example, between 0.962 and 1.357 (when sensitivity and specificity are 0.8 or higher).

[0075] The threshold can be obtained, for example, using a biological sample isolated from a healthy subject, a non-AD patient, an AD patient, a patient with amyloid-β accumulation, and / or a patient without amyloid-β accumulation (hereinafter also referred to as a "reference biological sample"). Furthermore, in the case of prognosis evaluation, for example, a reference biological sample isolated from the same subject after treatment may be used. The threshold may be measured, for example, simultaneously with the subject's sample, or may be measured in advance. The latter is preferable because, for example, it is not necessary to obtain a threshold each time the subject's sample is measured. It is preferable that the subject's sample and the reference biological sample are collected, for example, under the same conditions, and the ratio of the measured values ​​of the target phosphorylation sites is measured and calculated under the same conditions.

[0076] In the testing step, the possibility of the subject having Alzheimer's disease or the presence or absence of Alzheimer's disease can be appropriately determined depending on the ratio of the measured values ​​and the type of the threshold. Specifically, when the ratio of the measured values ​​is V2 / V1 and the threshold is used as a cut-off value, if V2 / V1 in the subject's sample is higher than the threshold, the subject can be evaluated as having, for example, a possibility of having AD, a high possibility of having AD, a high possibility of having AD, or having AD. On the other hand, if V2 / V1 in the subject's sample is equal to or lower than the threshold, the subject can be evaluated as having, for example, no possibility of having AD, a low possibility of having AD, a low possibility of having AD, or not having AD. When the ratio of the measured values ​​is V1 / V2 and the threshold value is used as a cut-off value, if V1 / V2 in the subject's sample is higher than the threshold value, the subject can be evaluated as, for example, not likely to have AD, low likely to have AD, low likely to have AD, or not having AD. On the other hand, if V1 / V2 in the subject's sample is equal to or lower than the threshold value, the subject can be evaluated as, for example, likely to have AD, high likely to have AD, or high likely to have AD, or having AD. The testing method of the present disclosure can also be applied, for example, to the preclinical AD stage (preclinical AD / preclinical stage of AD) in which AD-related pathological changes are present in the brain but are asymptomatic.

[0077] The evaluation method in the testing step has been described using the possibility of AD as an example, but the possibility or degree of progression may also be tested (evaluated) in the testing step. In this case, the degree of progression of AD can be evaluated by comparing the ratio of the measured values ​​in the subject's sample with the ratio of the measured values ​​in reference biological samples from AD patients at each stage of progression. Specifically, if the value of the subject's sample is similar to that of the reference biological samples at any stage of progression (if there is no significant difference), the subject can be evaluated as having a possibility of having AD at that stage of progression or as having AD at that stage of progression.

[0078] The testing step may involve evaluating the prognosis of the subject. When evaluating the prognosis of the subject in the testing step, for example, the evaluation may be performed in the same manner as described above, or the threshold value may be used as the threshold value to evaluate the prognosis. Specifically, when the ratio of the measured values ​​is V2 / V1 and the threshold value is used as the cut-off value, if the ratio of the measured values ​​in the subject's sample is higher than the threshold value, the subject can be evaluated as having an increase in phosphorylated tau fragments in samples such as body fluids after the treatment following the extraneuronal secretion of phosphorylated tau. Furthermore, the subject can be evaluated as having, for example, a suspicion (possibility) of relapse of AD, worsening of AD, or progression of AD. Furthermore, if the ratio of the measured values ​​in the subject's sample is equal to or less than the threshold value, the subject can be evaluated as having no or low suspicion (possibility) of relapse of AD, worsening of AD, or progression of AD after the treatment. When the ratio of the measured values ​​is V1 / V2 and the threshold is used as a cut-off value, if the ratio of the measured values ​​in the subject's sample is higher than the threshold, the subject can be evaluated as having no or low suspicion (possibility) of relapse of AD, worsening of AD, or progression of AD after the treatment. Furthermore, if the ratio of the measured values ​​in the subject's sample is equal to or lower than the threshold, the subject can be evaluated as having an increase in phosphorylated tau fragments in samples such as body fluids after the treatment, following extraneuronal secretion of phosphorylated tau. Furthermore, the subject can be evaluated as having, for example, suspicion (possibility) of relapse of AD, worsening of AD, or progression of AD.

[0079] In the testing method of the present disclosure, for example, samples from the same subject may be collected over time, and the ratio of the measured values ​​in the samples may be compared.Therefore, when the ratio of the measured values ​​is V2 / V1, for example, in the test step, if the ratio of the measured values ​​increases over time, it can be determined that the subject has AD, that the likelihood of AD has increased, or that AD has progressed due to the accumulation of Aβ and phosphorylated tau in the brain.In addition, when the ratio of the measured values ​​is V1 / V2, for example, in the test step, if the ratio of the measured values ​​decreases over time, it can be determined that the subject has AD, that the likelihood of AD has increased, or that AD has progressed.

[0080] The testing method of the present disclosure can be used, for example, to assist in determining, based on the evaluation step, whether to administer an AD disease-modifying drug, whether to continue administration of an AD disease-modifying drug, etc. Furthermore, the testing method of the present disclosure may include, for example, a step of administering a therapeutically effective amount of an AD disease-modifying drug to a subject who is evaluated in the evaluation step as having a possibility of, a high probability of, or being affected by AD.

[0081] The AD disease modifying drugs include, for example, anti-amyloid β antibodies, and specific examples include lecanemab (Rekembi (registered trademark)), donanemab, trontinemab, aducanumab, and the like.

[0082] The test method of the present disclosure may be combined with other known AD biomarkers. For example, it can be combined with the Aβ42 / 40 ratio. For example, by using a combination of multiple biomarkers, the test method of the present disclosure can more accurately predict the possibility of AD onset, the progression of AD, and the prognosis of a subject. [Example]

[0083] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0084] [Reference example 1] A second measurement reagent was prepared as follows, and phosphorylated tau peptide was measured.

[0085] (1) Preparation of alkaline phosphatase-labeled antibody dilution Monoclonal antibody A (antibody recognizing the amino acid sequence of tau 194-198) and monoclonal antibody B (antibody recognizing the amino acid sequence of tau 159-163) that specifically recognize tau protein were each digested with pepsin and purified by gel filtration to obtain F(ab')2 fragments. These fragments were then reduced, desalted, and mixed with maleimide-conjugated alkaline phosphatase (ALP) for coupling reaction. After quenching, desalting and gel filtration purification yielded the ALP-labeled antibodies. These were then diluted with 100 mM MOPS buffer (containing 1 mM MgCl2, 0.1 mM ZnCl2, 1% BSA, and 150 mM NaCl, pH 6.8) to obtain an ALP-labeled antibody dilution containing antibody A and antibody B.

[0086] (2) Measurement of phosphorylated tau peptides using the second assay reagent 150 μL of antibody-bound particle solution containing a monoclonal antibody against phosphorylated tau 181, included with Lumipulse (registered trademark) Phosphorylated Tau 181 (manufactured by Fujirebio Inc.), and 30 μL of the sample were dispensed into a reaction vessel, stirred, and then incubated at 37°C for 10 minutes. B / F separation and washing were then performed. For washing, "Lumipulse Washing Solution" (manufactured by Fujirebio Inc.) was used. After washing, 250 μL of the ALP-labeled antibody dilution solution prepared above was dispensed into the reaction vessel and incubated at 37°C for 10 minutes. B / F separation and washing were then performed. After washing, 200 μL of substrate solution (Lumipulse Substrate Solution, Fujirebio) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was dispensed into the reaction vessel, stirred, and allowed to react at 37°C for 5 minutes. The amount of luminescence was measured using a luminometer to obtain a count value. The actual measurement was performed using a fully automated chemiluminescent enzyme immunoassay system, Lumipulse G1200 (Fujirebio).

[0087] Similarly, a standard phosphorylated tau 181 solution was measured according to the protocol attached to the Lumipulse phosphorylated tau 181 kit, and a calibration curve was created based on the luminescence intensity. The concentration of tau protein phosphorylated at position 181 in each sample was calculated from the calibration curve.

[0088] [Example 1] We confirmed that AD patients (AD) and non-AD patients (non-AD) cases can be separated based on the ratio of measurements (second measurement: V2 / first measurement: V1) obtained using two assay systems with antibody combinations with different epitopes that measure tau protein phosphorylated at amino acid position 181 (pTau181). Non-AD includes patients with cognitive impairment due to causes other than AD (e.g., FTLD, CBD, DLB, etc.) and patients with other neurological diseases but normal cognitive function (e.g., psychiatric disorders, Parkinson's disease, etc.).

[0089] (1) Examination of the diagnostic ability of AD when using a physician's diagnosis as a reference Using a physician's diagnostic reference, the diagnostic ability of AD was examined by comparing the ratio of measured values ​​obtained with two assay systems using different antibody combinations targeting pTau181 epitopes. Specifically, 144 cerebrospinal fluid samples were measured using INNOTEST® PHOSPHO-TAU (181P) (Fujirebio Europe) (first assay reagent) and the second assay reagent described in Reference Example 1. The physician's diagnostic results in this case included 31 cases of Alzheimer's disease (including AD and MCI due to AD) and 113 cases of non-Alzheimer's disease (non-AD). The first assay reagent was used for ELISA assay according to the attached protocol. INNOTEST PHOSPHO-TAU (181P) is a reagent for measuring pTau181 by sandwich immunoassay using HT7 antibody (an antibody recognizing the amino acid sequence of tau 159-163) and AT270 antibody (pTau181 antibody). The second measurement reagent was measured according to Reference Example 1. After measurement using the first and second measurement reagents, the ratio (pTau ratio, V2 / V1) was calculated for the obtained values. Furthermore, as a reference example, the cerebrospinal fluid was measured using Lumipulse® β-Amyloid 1-42 (manufactured by Fujirebio Inc.) and Lumipulse® β-Amyloid 1-40 (manufactured by Fujirebio Inc.), and the Aβ42 / 40 ratio was calculated from the obtained values. Next, using the doctor's diagnosis as a reference, ROC analysis was performed on the measurement values ​​of the first measurement reagent, the measurement values ​​of the second measurement reagent, the ratio (V2 / V1) of the measurement values ​​of the first measurement reagent (V1) to the measurement values ​​of the second measurement reagent (V2), and the Aβ42 / 40 ratio. In the ROC analysis, the cut-off value for each item was calculated from the Youden Index, and the AUC (area under the ROC curve) values ​​for each item were compared. These results are shown in Figures 2 to 4 and Table 1 below.

[0090] Figure 2 is a graph showing the results of the ROC analysis. In Figure 2, the vertical axis represents the positive rate (sensitivity), and the horizontal axis represents the false positive rate (1-specificity). As shown in Figure 2, the pTau ratio (V2 / V1) calculated from the measured values ​​of the first measurement reagent and the second measurement reagent was closest to the upper left corner. These results demonstrate that the method using the pTau ratio (V2 / V1) has a high diagnostic ability for AD, and its diagnostic ability is higher than that of the commonly used Aβ42 / 40 ratio.

[0091] [Table 1]

[0092] Table 1 shows the cut-off value, AUC, and 95% CI (Confidence interval) for each measurement result. As shown in Table 1, the pTau ratio (V2 / V1) calculated from the measurement values ​​of the first measurement reagent and the second measurement reagent had a higher AUC and 95% CI than the Aβ42 / 40 ratio. These results show that the pTau ratio (V2 / V1) has a high diagnostic ability for AD when a reference based on a doctor's diagnosis is used.

[0093] Figure 3 is a graph (boxplot) showing the results of the Aβ42 / 40 ratio and pTau ratio in AD and non-AD. In Figure 3, (A) shows the results of the Aβ42 / 40 ratio in AD and non-AD, and (B) shows the results of the pTau ratio in AD and non-AD. In Figure 3(A), the vertical axis shows the Aβ42 / 40 ratio, and the horizontal axis shows the type of sample based on the diagnostic results of the specimen. In Figure 3(B), the vertical axis shows the pTau ratio, and the horizontal axis shows the type of sample based on the diagnostic results of the specimen. As shown in Figure 3, the dynamic range of the pTau ratio (V2 / V1) was approximately 20 times higher than that of the Aβ42 / 40 ratio. Therefore, it was found that the pTau ratio (V2 / V1) is easier to use in diagnosing AD than the Aβ42 / 40 ratio.

[0094] Figure 4 is a graph (boxplot) showing the results of the Aβ42 / 40 ratio and pTau ratio when AD and non-AD were further subdivided. In Figure 4, (A) shows the results of the Aβ42 / 40 ratio, and (B) shows the results of the pTau ratio. In Figure 4(A), the vertical axis shows the Aβ42 / 40 ratio, and the horizontal axis shows the sample type based on the subdivision of the specimen diagnostic results. In Figure 4(B), the vertical axis shows the pTau ratio, and the horizontal axis shows the sample type based on the subdivision of the specimen diagnostic results. In Figure 4, AD was subdivided into AD (dementia (dementia due to Alzheimer's disease)) and MCI due to AD (mild dementia due to Alzheimer's disease), and non-AD was subdivided into non-AD dementia (dementia other than Alzheimer's disease) and CU (cognitively unimpaired: cognitive function is normal). As shown in FIG. 4, it was found that even when AD and non-AD were further subdivided, AD could be diagnosed using the pTau ratio (V2 / V1).

[0095] (2) Examination of AD diagnostic ability when using amyloid PET results as a reference Using amyloid PET results as a reference, the AD diagnostic ability was examined by comparing the ratio of measurements obtained with two assay systems using different antibody combinations targeting pTau181 epitopes. Specifically, the same method as in Example 1(1) was used, except that amyloid PET results were used as the reference instead of the physician's diagnosis. These results are shown in Figure 5 and Table 2 below.

[0096] Figure 5 is a graph showing the results of the ROC analysis. In Figure 5, the vertical axis represents the positive rate (sensitivity), and the horizontal axis represents the false positive rate (1-specificity). As shown in Figure 5, the pTau ratio (V2 / V1) was close to the upper left corner, similar to the Aβ42 / 40 ratio. These results demonstrate that the method using the pTau ratio (V2 / V1) has a high diagnostic ability for AD, similar to the method using the Aβ42 / 40 ratio.

[0097] [Table 2]

[0098] Table 2 shows the cutoff value, AUC, and 95% CI (Confidence Interval) for each measurement result. As shown in Table 2, the pTau ratio was found to have a high AUC and 95% CI, similar to the Aβ42 / 40 ratio. These results indicate that when amyloid PET results are used as a reference, the pTau ratio (V2 / V1) has a high diagnostic ability for AD, comparable to the Aβ42 / 40 ratio. These results suggest that the pTau ratio (V2 / V1) has similar diagnostic ability for AD as amyloid PET, and is therefore expected to be used as a substitute for amyloid PET for diagnosing AD.

[0099] From the above results, it was considered that the pTau ratio (V2 / V1) could be used to grasp the accumulation of amyloid in the brain and at the same time to grasp the progression of neurodegeneration. Therefore, the correlation between the pTau ratio (V2 / V1) and the score of the Mini-Mental State Examination (MMSE), a cognitive function test, was examined using 95 subjects (144 cases) in Example 1 (1) who had data on MMSE. The correlation coefficient was r = -0.565 (p < 0.001), r 2 = 0.319. For reference, when the correlation between the Aβ42 / 40 ratio and the MMSE score was examined, the correlation coefficient was r = 0.399 (p < 0.001), r 2 = 0.159. Therefore, the pTau ratio (V2 / V1) can be said to be an indicator that shows a higher correlation with the cognitive function of the subject compared to the Aβ42 / 40 ratio.

[0100] In addition, AD testing using the Aβ42 / 40 ratio in cerebrospinal fluid can determine whether amyloid accumulation exists in the brain, but it has the drawback of not being able to determine the extent of accumulation or the degree of neurodegeneration. On the other hand, the pTau ratio (V2 / V1) shows a high correlation with MMSE scores, so it is expected to be able to evaluate not only the accumulation of amyloid beta but also the progression of neurodegeneration (degree of cognitive decline).

[0101] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0102] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Alzheimer's disease testing methods> (Appendix 1) a measuring step of measuring the amount of target phosphorylated sites in two or more types of peptide fragments derived from phosphorylated tau protein in a target sample using two or more measurement systems; a comparison step of comparing the magnitudes of the measured values ​​of the target phosphorylation portion, The measuring step a first measurement step of measuring the amount of the target phosphorylated site using a first measurement system comprising a first binding molecule that binds to the target phosphorylated site and a second binding molecule that binds to the non-phosphorylated site of the peptide fragment; a second measurement step of measuring the amount of the target phosphorylated site using a second measurement system comprising a third binding molecule that binds to the target phosphorylated site and a fourth binding molecule that binds to the non-phosphorylated site of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; A method for testing Alzheimer's disease, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is shared by the two or more types of peptide fragments. (Appendix 2) The phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The threonine residue is selected from the group consisting of threonine residues at positions 69, 71, 123, 153, 175, 181, 205, 212, 217, 231, 403, 414, and 427 in tau protein. a phosphorylated serine residue in the phosphorylated tau protein, the serine residue is selected from the group consisting of serine residues at positions 46, 68, 113, 184, 185, 191, 198, 199, 202, 208, 210, 214, 235, 237, 238, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416, 422, 433, and 435 in the tau protein; and / or a phosphorylated tyrosine residue in the phosphorylated tau protein, The tyrosine residue is a tyrosine residue selected from the group consisting of tyrosine residues at positions 18, 197, and 394 in tau protein. Testing method described in Appendix 1. (Appendix 3) the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The testing method according to Appendix 1, wherein the threonine residue is the threonine residue at positions 181, 212, 217, and / or 231 in tau protein. (Appendix 4) the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The testing method according to Appendix 1, wherein the threonine residue is the threonine residue at position 181 in tau protein. (Appendix 5) The testing method according to Appendix 4, wherein the non-phosphorylated portion to which the second binding molecule binds is a peptide region from positions 1 to 180 in tau protein. (Appendix 6) The testing method according to Appendix 4 or 5, wherein the non-phosphorylated portion to which the second binding molecule binds is a peptide region at positions 1 to 180 in the tau protein, and the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region at positions 182 to 441 in the tau protein. (Appendix 7) 7. The testing method according to any one of appendices 1 to 6, comprising a testing step of testing the possibility of the subject suffering from Alzheimer's disease based on the magnitude relationship between the measured values ​​of the target phosphorylation sites. (Appendix 8) The testing method described in Appendix 7, wherein the magnitude relationship between the measurement values ​​of the target phosphorylation sites is a ratio of the measurement values ​​of the target phosphorylation sites or a difference in the measurement values ​​of the target phosphorylation sites. (Appendix 9) The testing method described in Appendix 8, comprising a testing step of comparing the ratio or difference of the measured value of the target phosphorylation portion with a threshold value to test the possibility of the subject suffering from Alzheimer's disease. (Appendix 10) The testing method according to any one of appendices 1 to 9, wherein the comparison of the magnitudes of the measured values ​​is a calculation of the ratio or difference of the measured values ​​of the target phosphorylation site. (Appendix 11) The test method according to any one of Appendices 8 to 10, wherein the ratio of the measurement value of the target phosphorylation portion is the ratio (V2 / V1) of the measurement value (V2) obtained in the second measurement step to the measurement value (V1) obtained in the first measurement step. (Appendix 12) The first measuring step a first complex formation step of contacting the subject sample with the first binding molecule and the second binding molecule to form a first complex between the peptide fragment containing the target phosphorylation site in the subject sample and the first binding molecule and the second binding molecule; and a first complex measurement step of measuring the amount of target phosphorylated sites in the sample by detecting the first complex. (Appendix 13) The first complex formation step includes: contacting the subject sample with the second binding molecule to form a complex between the peptide fragment containing the target phosphorylation site in the subject sample and the second binding molecule; The testing method according to claim 12, wherein the complex is contacted with the first binding molecule to form the first complex. (Appendix 14) the first binding molecule comprises a label; The testing method according to claim 13, wherein the first complex measuring step measures the amount of target phosphorylated sites in the sample by detecting the label in the first complex. (Appendix 15) The second measuring step includes: a second complex formation step of contacting the subject sample with the third binding molecule and the fourth binding molecule to form a second complex between the peptide fragment containing the target phosphorylation site in the subject sample and the third binding molecule and the fourth binding molecule; and a second complex measurement step of measuring the amount of target phosphorylated sites in the sample by detecting the second complex. (Appendix 16) The second complex formation step comprises: contacting the subject sample with the third binding molecule to form a complex between the peptide fragment containing the target phosphorylation site in the subject sample and the third binding molecule; The testing method according to claim 15, wherein the complex is contacted with the fourth binding molecule to form the second complex. (Appendix 17) the fourth binding molecule comprises a label; The testing method according to claim 16, wherein the second complex measuring step measures the amount of target phosphorylated sites in the sample by detecting the label in the second complex. (Appendix 18) 18. The testing method of any one of claims 1 to 17, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule is an antibody. (Appendix 19) 19. The testing method of any one of appendices 1 to 18, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule have a label. (Appendix 20) 20. The testing method according to any one of claims 1 to 19, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule has a carrier. (Appendix 21) the first binding molecule comprises a label; 21. The method of any one of claims 1 to 20, wherein the second binding molecule comprises a carrier. (Appendix 22) the third binding molecule comprises a carrier; 22. The testing method of any one of claims 1 to 21, wherein the fourth binding molecule has a label. (Appendix 23) 23. The testing method of any one of claims 1 to 22, wherein the sample comprises a blood sample or cerebrospinal fluid. <Alzheimer's disease test kit> (Appendix 24) two or more measurement reagents for use in measuring the amount of target phosphorylated sites in two or more peptide fragments derived from phosphorylated tau protein in a subject sample; The first measurement reagent is a first binding molecule that binds to the target phosphorylation site; a second binding molecule that binds to the non-phosphorylated portion of the peptide fragment; The second measurement reagent is a third binding molecule that binds to the target phosphorylation site; a fourth binding molecule that binds to the non-phosphorylated portion of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; a test kit for Alzheimer's disease, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is shared by the two or more types of peptide fragments; (Appendix 25) The phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The threonine residue is selected from the group consisting of threonine residues at positions 69, 71, 123, 153, 175, 181, 205, 212, 217, 231, 403, 414, and 427 in tau protein. a phosphorylated serine residue in the phosphorylated tau protein, the serine residue is selected from the group consisting of serine residues at positions 46, 68, 113, 184, 185, 191, 198, 199, 202, 208, 210, 214, 235, 237, 238, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416, 422, 433, and 435 in the tau protein; and / or a phosphorylated tyrosine residue in the phosphorylated tau protein, The tyrosine residue is a tyrosine residue selected from the group consisting of tyrosine residues at positions 18, 197, and 394 in tau protein. 2. The test kit described in Appendix 24. (Appendix 26) the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, 26. The test kit according to claim 24 or 25, wherein the threonine residue is the threonine residue at position 181, 212, 217, and / or 231 in tau protein. (Appendix 27) the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, 27. The test kit according to any one of Appendices 24 to 26, wherein the threonine residue is the threonine residue at position 181 in tau protein. (Appendix 28) 28. The test kit according to claim 27, wherein the non-phosphorylated portion to which the second binding molecule binds is a peptide region from positions 1 to 180 in the phosphorylated tau protein. (Appendix 29) 29. The test kit according to claim 27 or 28, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region from positions 182 to 441 in the phosphorylated tau protein. (Appendix 30) 30. The test kit of any of claims 24 to 29, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule is an antibody. (Appendix 31) 31. The test kit of any of claims 24 to 30, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule comprises a label. (Appendix 32) 32. The test kit of any one of claims 24 to 31, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule comprises a carrier. (Appendix 33) the first binding molecule comprises a label; 33. The test kit of any of claims 24 to 32, wherein the second binding molecule comprises a carrier. (Appendix 34) the third binding molecule comprises a carrier; 34. The test kit of any of claims 24 to 33, wherein the fourth binding molecule comprises a label. (Appendix 35) A test kit according to any one of appendices 24 to 34 for use in the test method according to any one of appendices 1 to 21. (Appendix 36) 36. The test kit of any of claims 24 to 35, wherein the sample comprises a blood sample or cerebrospinal fluid. [Industrial Applicability]

[0103] As described above, the present disclosure provides a new method that can be used to test for Alzheimer's disease and a test kit that can be used for the method. Therefore, the present disclosure is extremely useful, for example, in the field of testing.

Claims

1. a measuring step of measuring the amount of target phosphorylated sites in two or more types of peptide fragments derived from phosphorylated tau protein in a subject sample using two or more measurement systems; a comparison step of comparing the magnitudes of the measured values ​​of the target phosphorylation portion, The measuring step a first measurement step of measuring the amount of the target phosphorylated site using a first measurement system comprising a first binding molecule that binds to the target phosphorylated site and a second binding molecule that binds to the non-phosphorylated site of the peptide fragment; a second measurement step of measuring the amount of the target phosphorylated site using a second measurement system comprising a third binding molecule that binds to the target phosphorylated site and a fourth binding molecule that binds to a non-phosphorylated site of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; A method for testing for Alzheimer's disease, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is common to the two or more types of peptide fragments.

2. The phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, the threonine residue is selected from the group consisting of positions 69, 71, 123, 153, 175, 181, 205, 212, 217, 231, 403, 414, and 427 in tau protein; a phosphorylated serine residue in the phosphorylated tau protein, the serine residue is selected from the group consisting of serine residues at positions 46, 68, 113, 184, 185, 191, 198, 199, 202, 208, 210, 214, 235, 237, 238, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416, 422, 433, and 435 in the tau protein; and / or a phosphorylated tyrosine residue in the phosphorylated tau protein, The method of claim 1, wherein the tyrosine residue is a tyrosine residue selected from the group consisting of tyrosine residues at positions 18, 197, and 394 in tau protein.

3. the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The method of claim 1 , wherein the threonine residue is the threonine residue at position 181, 212, 217, and / or 231 of tau protein.

4. the phosphorylated portion is a phosphorylated threonine residue in the phosphorylated tau protein, The method of claim 1 , wherein the threonine residue is the threonine residue at position 181 in tau protein.

5. The method of claim 4, wherein the non-phosphorylated portion to which the second binding molecule binds is a peptide region from positions 1 to 180 in tau protein.

6. the non-phosphorylated portion to which the second binding molecule binds is a peptide region from positions 1 to 180 in tau protein; The testing method according to claim 4, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region from positions 182 to 441 in tau protein.

7. The testing method according to claim 1 or 2, further comprising a testing step of testing the possibility of the subject suffering from Alzheimer's disease based on the magnitude correlation between the measured values ​​of the target phosphorylation sites.

8. The testing method according to claim 1 or 2, wherein the comparison of the magnitudes of the measured values ​​is a calculation of a ratio of the measured values ​​of the target phosphorylation sites.

9. The test method according to claim 8, wherein the ratio of the measurement values ​​of the target phosphorylation portion is the ratio (V2 / V1) of the measurement value (V2) obtained in the second measurement step to the measurement value (V1) obtained in the first measurement step.

10. The first measuring step a first complex formation step of contacting the subject sample with the first binding molecule and the second binding molecule to form a first complex between the peptide fragment containing the target phosphorylation site in the subject sample and the first binding molecule and the second binding molecule; a first complex measuring step of measuring the amount of target phosphorylated sites in the sample by detecting the first complex; The second measuring step a second complex formation step of contacting the subject sample with the third binding molecule and the fourth binding molecule to form a second complex between the peptide fragment containing the target phosphorylation site in the subject sample and the third binding molecule and the fourth binding molecule; The testing method according to claim 1 or 2, further comprising a second complex measuring step of measuring the amount of target phosphorylated sites in the sample by detecting the second complex.

11. The testing method according to claim 1 or 2, wherein the first binding molecule, the second binding molecule, the third binding molecule, and / or the fourth binding molecule is an antibody.

12. The testing method according to claim 1 or 2, wherein the sample comprises a blood sample or cerebrospinal fluid.

13. two or more measurement reagents for use in measuring the amount of target phosphorylated sites in two or more types of peptide fragments derived from phosphorylated tau protein in a subject sample; The first measurement reagent is a first binding molecule that binds to the target phosphorylation site; a second binding molecule that binds to the non-phosphorylated portion of the peptide fragment; The second measurement reagent is a third binding molecule that binds to the target phosphorylation site; a fourth binding molecule that binds to the non-phosphorylated portion of the peptide fragment; the non-phosphorylated portion to which the second binding molecule binds is a peptide region that is not present in at least one of the two or more peptide fragments; A test kit for Alzheimer's disease, wherein the non-phosphorylated portion to which the fourth binding molecule binds is a peptide region that is common to the two or more types of peptide fragments.

14. The test kit according to claim 13, for use in the test method according to claim 1 or 2.

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