Method for measuring phosphorylated tau protein, and kit for measuring phosphorylated tau protein

Abstract

Provided are a measurement method and a measurement kit which make it possible to suppress an albumin-dependent increase in background. A method for measuring phosphorylated tau protein according to the present disclosure includes a measurement step which includes: bringing a target sample into contact with a measurement reagent for phosphorylated tau protein in the presence of albumin and an amino group-containing compound, wherein the phosphorylated tau protein has a structure such that a threonine residue at position-217 is phosphorylated; and measuring the phosphorylated tau protein, which has a structure such that the threonine residue at position-217 is phosphorylated, in the sample.

Description

Method for measuring phosphorylated tau protein and kit for measuring phosphorylated tau protein

[0001] The present disclosure relates to a method for measuring phosphorylated tau protein and a kit for measuring phosphorylated tau protein.

[0002] Alzheimer's disease (AD) is considered to be a continuous disease, similar to lifestyle-related diseases. 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] 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.

[0004] The present inventors have found a problem that, when attempting to measure phosphorylated tau protein in which the threonine residue at position 217 (hereinafter also referred to as "pTau217") by immunoassay, the measured value may be significantly increased and the signal-to-noise (S / N) ratio may be reduced in samples that substantially do not contain pTau217, i.e., the background may be significantly increased and the S / N ratio may be reduced. This problem is not observed in immunoassays of phosphorylated tau protein in which other phosphorylation sites are phosphorylated, and it was presumed to be a phenomenon specific to the threonine residue at position 217. The present inventors have found that the increase in background is due to differences in the albumin contained in the assay reagent.

[0005] Therefore, an object of the present disclosure is to provide a measurement method that can suppress an increase in background caused by albumin, particularly bovine serum albumin, and a measurement kit that can be used for said method.

[0006] In order to achieve the above-mentioned object, the method for measuring phosphorylated tau protein (hereinafter also referred to as "measurement method") of the present disclosure includes a measurement step of contacting a subject sample with a measurement reagent for phosphorylated tau protein in which threonine at position 217 is phosphorylated in the presence of albumin and an amino group-containing compound, and measuring phosphorylated tau protein in the sample in which threonine at position 217 is phosphorylated.

[0007] The assay kit for phosphorylated tau protein (hereinafter also referred to as "assessment kit") of the present disclosure comprises an amino group-containing compound, albumin, and an assay reagent for phosphorylated tau protein in which threonine at position 217 is phosphorylated.

[0008] According to the present disclosure, it is possible to provide a measurement method that can suppress an increase in background caused by albumin, particularly bovine serum albumin, and a measurement kit that can be used for said method.

[0009] FIG. 1 shows the mechanism of background estimation by albumin, where (A) is a schematic diagram showing the measurement state in the presence of albumin but in the absence of an amino group-containing compound, and (B) is a schematic diagram showing the measurement state in the presence of albumin and an amino group-containing compound.

[0010] <Definitions> As used herein, "tau protein" (hereinafter also referred to as "tau") refers to a protein that is mainly expressed 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.

[0011] Tau protein (SEQ ID NO: 1, NP_005901.2) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPT REPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0012] As used herein, "phosphorylated tau protein" (hereinafter also referred to as "phosphorylated tau") refers to tau in which at least one amino acid residue is phosphorylated. The phosphorylated amino acid residue is, for example, a threonine residue at positions 175, 181, 184, 205, 212, 217, and / or 231 in the tau, and / or a serine residue at positions 185, 191, 198, 199, 202, 208, 214, 235, 262, 356, 396, 404, 409, 416, and / or 422 in the tau. The threonine residue at positions 181 and / or 217 is known to be more highly associated with Alzheimer's disease. As used herein, "phosphorylated tau protein phosphorylated at threonine at position 217" (hereinafter also referred to as "pTau217") is defined as tau protein phosphorylated at at least threonine at position 217, and may further be phosphorylated at one or more other amino acid residues. The phosphorylation of tau can be detected, for example, using a binding molecule that binds to the target phosphorylated amino acid residue.

[0013] As used herein, "amino group-containing compound" means any compound that contains a primary amine, i.e., any compound that contains an amino group.

[0014] 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, sheep, 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 suffering from 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 checkup. Examples of clinical findings include findings based on cognitive function tests, brain MRI images, etc.

[0015] As used herein, a "sample" may contain phosphorylated tau or may potentially contain phosphorylated tau. Examples of the sample include biological samples such as 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 solution, Tricine buffer solution, etc.), Tris buffer solution, phosphate buffer solution, and glycine buffer solution. Furthermore, the sample may be diluted, pretreated, or the like prior to measurement.

[0016] 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 include instructions or instructions describing how to use the test reagent, diagnostic reagent, etc.

[0017] 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, include 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 website provided on the Internet, email, etc.).

[0018] 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 or more amino acids.

[0019] 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.

[0020] 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; and the like.

[0021] 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, sugar chains, and the like capable of binding to the predetermined molecule. Specific examples of the binding molecule include aptamers, antibodies, receptors, ligands, and the like 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 antibodies may be derived from animals such as mammals, including mice, rats, hamsters, rabbits, sheep, goats, cattle, horses, camels, and alpacas; birds, including chickens and ostriches; and cartilaginous fish, including sharks. Examples of the antibodies include camelid-derived heavy chain antibodies (VHH antibodies), cartilaginous fish-derived immunoglobulin new antigen receptors (IgNARs), and antibody fragments (e.g., Fab, Fab', and F(ab')). ２ , single domain antibodies (nanobodies), etc.), recombinant antibodies (e.g., scFv, disulfide-linked Fv (dsFv), diabodies, minibodies, etc.). The antibody may also be an antibody-like molecule (e.g., affibody, anticalin, DARPins, monobody, etc.) produced by molecular biological techniques such as phage display and / or by protein engineering techniques using existing protein motifs.

[0022] 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. Furthermore, the nucleic acid sequence of RNA can also be obtained from the corresponding DNA base sequence using appropriate sequence conversion software, etc.

[0023] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0024] <Tau Protein Measurement Kit> In one aspect, the present disclosure provides a kit capable of suppressing an increase in background signal caused by albumin, particularly bovine serum albumin. The tau protein measurement kit of the present disclosure includes an amino group-containing compound, albumin, and a measurement reagent for phosphorylated tau protein (pTau217) in which threonine at position 217 is phosphorylated.

[0025] The present inventors have discovered a problem in immunoassays for measuring pTau217 in samples substantially free of pTau217: the measured value may be significantly elevated and the signal-to-noise (S / N) ratio may be reduced, i.e., the background may be significantly elevated and the S / N ratio may be reduced. This problem is not observed in immunoassays for phosphorylated tau protein phosphorylated at other phosphorylation sites, and was presumed to be a phenomenon specific to the threonine residue at position 217. After extensive research, the present inventors have discovered that the background elevation is due to differences in the albumin contained in the assay reagent, and that the albumin-induced background elevation can be suppressed by an amino group-containing compound, leading to the establishment of the present disclosure. Therefore, the present disclosure can suppress the background elevation caused by albumin, particularly bovine serum albumin. Furthermore, the present disclosure can suppress the albumin-induced background elevation, thereby improving the S / N ratio in assay systems containing albumin. Since the measurement method of the present disclosure can suppress the background increase caused by albumin in the measurement of phosphorylated tau protein, it can also be said to be, for example, a kit for suppressing the background increase caused by albumin in the measurement of phosphorylated tau protein.

[0026] The background increase caused by albumin and the suppression of the background increase by the amino group-containing compound are presumed to be due to the following mechanism. However, the present disclosure is not limited by the following presumption. Albumin is a protein commonly added as a blocking agent to measurement reagents used in immunoassays, and has the property of adsorbing various substances. Therefore, as shown in FIG. 1(A), some albumin is presumed to adsorb a measurement inhibitory substance X that can bind to the measurement reagent used in the immunoassay of pTau217. When the albumin used in the measurement reagent adsorbs measurement inhibitory substance X, binding between the measurement reagent and measurement inhibitory substance X occurs independently of phosphorylated tau protein during the measurement, resulting in an increase in background. Furthermore, the adsorption amount of measurement inhibitory substance X is presumed to vary depending on the production lot of albumin used. Therefore, it is presumed that the amount of measurement inhibitory substance X adsorbed by albumin varies depending on the albumin used in the measurement reagent, resulting in a difference between a large background increase and a case where no background increase is observed. On the other hand, the amino group-containing compound is presumed to bind to the measurement inhibitory substance X via its amino group, as shown in Figure 1 (B) . Therefore, since the measurement kit of the present disclosure contains the amino group-containing compound, even when the albumin contains the measurement inhibitory substance X, the amino group-containing compound binds to the measurement inhibitory substance X, thereby suppressing the binding between the measurement reagent and the measurement inhibitory substance X, and it is therefore presumed that an increase in background caused by albumin can be suppressed.

[0027] The albumin can be of any origin as long as it is usable as a blocking agent in immunoassays, and a specific example is serum albumin. The serum albumin is preferably derived from an animal. Examples of the animal include humans, cows, mice, rats, hamsters, rabbits, sheep, goats, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions, and preferably cows or horses. The amino group-containing compound can, for example, particularly suitably suppress an increase in background caused by bovine serum albumin.

[0028] Examples of the amino group-containing compound include tris(hydroxymethyl)aminomethane (Tris), amino acids, ethanolamine, methylamine, polymers containing primary amines, and proteins such as casein (excluding albumin). Examples of the amino acid include glycine, valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, and serine. Glycine and histidine are preferred, and glycine is more preferred, because they can effectively suppress an increase in background. The amino group-containing compound may include one or more types. When the kit of the present disclosure includes multiple types of amino group-containing compounds, the amino group-containing compounds are preferably Tris and an amino acid, and more preferably Tris and glycine, because they can effectively suppress an increase in background. When the amino group-containing compound is a buffer such as Tris, the amino group-containing compound may be used as a buffer.

[0029] Hereinafter, an example will be described in which the first binding molecule is a binding molecule that binds to pTau217 and the second binding molecule binds to the tau protein, but the binding properties of the first binding molecule and the second binding molecule may be reversed. In this case, in the following description, the "first binding molecule" can be read as the "second binding molecule" and the "second binding molecule" can be read as the "first binding molecule," and the description therein can be used.

[0030] The pTau217 assay reagent is a reagent capable of specifically detecting or measuring pTau217, and includes, for example, a first binding molecule for pTau217. The first binding molecule may, for example, recognize and bind only to the phosphorylated threonine acid residue at position 217 in pTau217, or may recognize and bind to a peptide region containing the phosphorylated threonine acid residue at position 217, i.e., the phosphorylated amino acid residue at position 217 and its surrounding peptide region. The first binding molecule preferably specifically binds to pTau217. The first binding molecule is preferably an antibody or an antigen-binding fragment thereof that specifically binds to pTau217. The antibody against pTau217 can be produced by appropriately adopting and improving a conventionally known method. When the antibody against pTau217 is, for example, a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495-497, 1975). Furthermore, the antibody against pTau217 is commercially available, and commercially available antibodies can also be used. Specific 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).

[0031] The first binding molecule for pTau217 may be supported on a carrier. That is, the pTau217 measurement reagent may contain a carrier supporting the first binding molecule for pTau217. 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. The binding molecule may be provided in a form impregnated in a medium such as filter paper. The first binding molecule for pTau217 may be configured to be supported on a carrier during pTau217 measurement. That is, in the kit of the present disclosure, the pTau217 measurement reagent may be configured so that the first binding molecule for pTau217 is supported on the carrier by being present in a separate state and coexisting during measurement. In this case, the kit of the present disclosure may comprise, for example, binding a first component of a pair of affinity substances to a first binding molecule for pTau217, binding a second component of the pair of affinity substances to the carrier, and supporting the first binding molecule for pTau217 on the carrier via affinity binding between the first and second components. The pair of affinity substances comprises 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 may also be referred to as, for example, a tag and a binding partner.

[0032] The method for immobilizing the first binding molecule on the carrier can be a conventionally known method or a method similar thereto, depending on the type of the first binding molecule. The first binding molecule may be immobilized directly or indirectly on the surface of the carrier. Examples of direct immobilization methods 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 the first binding molecule is immobilized by binding the first binding molecule to the carrier via a covalent bond formed by reaction between the active group and the first binding molecule. Examples of indirect immobilization methods 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. The first binding molecule may be supported on the carrier by ionic bonding with the carrier or by being adsorbed to the carrier. When the first binding molecule for pTau217 is indirectly supported on the carrier, for example, the antibody for pTau217 and the carrier may be contained in the same container or in separate containers.

[0033] The pTau217 measurement reagent may include, for example, a second binding molecule for the (phosphorylated) tau protein or a complex of the pTau217 and the first binding molecule. In this case, the second binding molecule may be configured so as to be capable of measuring the phosphorylation of threonine at position 217 in phosphorylated tau protein when used in combination with the first binding molecule. That is, the second binding molecule is preferably configured so as not to compete with the first binding molecule for binding to pTau217. The second binding molecule may recognize, for example, a phosphorylated amino acid residue other than position 217 in the phosphorylated tau protein or a region containing the amino acid residue (hereinafter collectively referred to as a "phosphorylated region"), or may recognize an unphosphorylated region in the phosphorylated tau protein (hereinafter also referred to as a "non-phosphorylated region"). The second binding molecule may include both a binding molecule that recognizes the phosphorylated region and a binding molecule that recognizes the non-phosphorylated region.

[0034] When the second binding molecule binds to the phosphorylated region, the second binding molecule is preferably an antibody or an antigen-binding fragment thereof that specifically binds to the phosphorylated region. For example, when the second binding molecule is a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495-497, 1975). Antibodies that specifically bind to the phosphorylated region are commercially available, and commercially available antibodies can also be used. Specific 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), and polyclonal antibody (AB9652, Merck). Examples of antibodies that bind to the phosphorylated serine residue (position 202 or 205) include AT8 (Fujirebio Europe). Examples of antibodies that bind to the phosphorylated threonine residue (position 212 or 214) include AT100 (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 the antibody that binds to the phosphorylated serine residue (position 396) include PHF13.6 (manufactured by Thermo Fisher Scientific) and 5HCLC (manufactured by Thermo Fisher Scientific). Examples of the antibody that binds to the phosphorylated serine residue (position 396 or 404) include PFH1 (manufactured by Creative Biolabs). Examples of the antibody that binds to the phosphorylated serine residue (position 416) include the antibodies described in WO 2013 / 180238.

[0035] When the second binding molecule binds to the non-phosphorylated region, the non-phosphorylated region to which the second binding molecule binds is, for example, the peptide region of positions 1 to 216 or 218 to 441 of the tau protein, preferably the peptide region of positions 1 to 210 or 220 to 441, more preferably the peptide region of positions 1 to 175, 185 to 210, or 220 to 441, and particularly preferably the peptide region of positions 159 to 163 or 194 to 198. The second binding molecule may comprise two or more types of binding molecules. When the second binding molecule comprises two types of binding molecules, the non-phosphorylated regions to which the second binding molecules bind are, for example, positions 1 to 175 and 185 to 210, positions 1 to 175 and 220 to 441, positions 185 to 210 and 220 to 441, and preferably positions 159 to 163 and 194 to 198, respectively.

[0036] The second binding molecule is preferably an antibody or an antigen-binding fragment thereof that specifically binds to the non-phosphorylated region. For example, if the second binding molecule is a monoclonal antibody, it can be produced by a method for producing monoclonal antibodies using hybridomas (Kohler & Milstein, Nature, 256:495, 1975). Antibodies that specifically bind to the non-phosphorylated region are commercially available, and commercially available antibodies can also be used. Examples of the second binding molecule include LRL (Eli Lilly and Company, epitope: positions 111 to 130), 4G10-E2 (Eli Lilly and Company, epitope: positions 111 to 130), ADx204 (ADx NeuroSciences, epitope: positions 6 to 18), Tau12 (Quanterix, epitope: positions 6 to 18), HT7 (Thermo Fisher, epitope: positions 159 to 163), and HT43 (Janssen, epitope: positions 7 to 20).

[0037] The reagent for measuring pTau217 containing the first binding molecule and the second binding molecule may be, for example, a commercially available kit, such as Lumipulse (registered trademark) G pTau217 Immunoreaction Cartridges (manufactured by Fujirebio Europe).

[0038] The first binding molecule and / or the second binding molecule may have a label. In this case, pTau217 can be detected via the first binding molecule and / or the second binding molecule by detecting the label. When the pTau217 assay reagent contains the first binding molecule and the second binding molecule, it is preferable that one of the first binding molecule and the second binding molecule is supported on the carrier, and the other is labeled. It is more preferable that the first binding molecule is supported on the carrier, and the second binding molecule is labeled. The method for introducing a label into the first binding molecule and / or the second binding molecule can be carried out by appropriately adopting a conventionally known method or a method equivalent thereto, depending on the type of the first binding molecule and the second 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 the first binding molecule on the carrier.

[0039] When the first binding molecule and / or the second binding molecule has a label, the kit of the present disclosure may also 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, it can also be referred to as a substrate liquid.

[0040] When the measurement reagent contains the first binding molecule and the second binding molecule, the first binding molecule and the second binding molecule may be contained in a mixed state or may be contained separately in the measurement reagent. In the measurement reagent, the first binding molecule and the second binding molecule may each be composed of one or more types.

[0041] The first binding molecule and / or the second binding molecule of the assay 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; and the like, with Tris buffer being preferred. 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 value 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, a chelating agent such as EDTA, a sugar such as sucrose, or a preservative such as sodium azide.

[0042] The assay kit of the present disclosure may further include a pTau217 standard. The pTau217 standard is an aqueous solution containing pTau217 at one or more predetermined concentrations, or a pTau217 powder (e.g., a lyophilized product). The pTau217 standard is useful, for example, as a control. Furthermore, by using the pTau217 standard, for example, in the pTau217 assay method described below, a calibration curve corresponding to the pTau217 concentration can be prepared, and the concentration of pTau217 in a sample can be analyzed.

[0043] The measurement kit of the present disclosure may include, for example, a diluent (first diluent) for diluting the first binding molecule, a diluent (second diluent) for diluting the second binding molecule, a washing solution for washing the first complex or the second complex formed by the reaction between the sample and the first binding molecule or the second binding molecule, a sample pretreatment solution (treatment solution), etc.

[0044] The diluent contains, for example, the buffer solution. The cleaning solution contains, for example, the buffer solution and a nonionic surfactant. The diluent and / or the cleaning solution may contain, for example, the albumin and / or the amino group-containing compound.

[0045] In the measurement reagent of the present disclosure, for example, the albumin and the amino group-containing compound are arranged so that both the albumin and the amino group-containing compound coexist during the reaction between the sample (pTau217 in the sample) and the first binding molecule and / or the second binding molecule, which can effectively suppress an increase in background. Therefore, preferably, the albumin and the amino group-containing compound are arranged so that both the albumin and the amino group-containing compound coexist during the reaction between the sample (pTau217 in the sample) and the first binding molecule and during the reaction between the sample (pTau217 in the sample) and the second binding molecule. Furthermore, in the binding molecule of the present disclosure, it is preferable that the albumin is configured to contact the first binding molecule and / or the second binding molecule during the reaction between the sample (pTau217 in the sample) and the first binding molecule and / or the second binding molecule. As a specific example, in the measurement reagent of the present disclosure, the first binding molecule and / or the second binding molecule may comprise the albumin and the amino group-containing compound. When the measurement reagent of the present disclosure includes the first diluent, the first diluent may comprise the albumin and the amino group-containing compound. When the measurement reagent of the present disclosure includes the first diluent, the first binding molecule may comprise the albumin and a portion of the amino group-containing compound, and the first diluent may comprise the remainder of the albumin and the amino group-containing compound. When the measurement reagent of the present disclosure includes the second diluent, the second diluent may comprise the albumin and the amino group-containing compound. When the measurement reagent of the present disclosure includes the second diluent, the second binding molecule may comprise the albumin and a portion of the amino group-containing compound, and the second diluent may comprise the remainder of the albumin and the amino group-containing compound. When the measurement reagent of the present disclosure includes a treatment solution, the treatment solution may comprise the albumin and the amino group-containing compound.

[0046] In the measurement 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.

[0047] In the measurement 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 measurement kit of the present disclosure, when all reagents are contained in a single container in a mixed or unmixed state, the measurement kit of the present disclosure can also be referred to as, for example, a measurement reagent for phosphorylated tau protein.

[0048] The measurement kit of the present disclosure may further include, for example, a container for storing the components of the kit. In this case, the measurement kit of the present disclosure may be provided with each component contained in a different container (e.g., a tube, a plate, etc.). The measurement 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 pTau217. Examples of the device structure include: 1) a device having a first area for mixing a sample with either the first binding molecule or the second binding molecule to prepare a mixture, and a second area for contacting the prepared mixture with the other binding molecule of the first binding molecule or the second binding molecule to detect the pTau217; 2) a device having an area for mixing a sample with the first binding molecule or the second binding molecule to detect the pTau217; 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 pTau217.

[0049] The measurement kit of the present disclosure may include, for example, instructions or manuals.

[0050] The assay kit of the present disclosure can suppress the albumin-dependent background increase by the amino group-containing compound, for example. The suppression of the background increase can be evaluated, for example, according to Example 1 described below, using an assay system containing the target amino group-containing compound and bovine serum albumin, and a control assay system that is similar except that the target amino group-containing compound is not added. Specifically, in the evaluation, for example, first, each assay system is prepared by adding each reagent to the assay system containing the first binding molecule and the second binding molecule so that the bovine serum albumin concentration is 1.11 (w / v)% and the target amino group-containing compound concentration is 27.78 mM, and then measurements (e.g., luminescence intensity or count values) are obtained for each assay system. When the measured value (T) of the measurement system containing the target amino group-containing compound and bovine serum albumin is lower than the measured value (C) of the control measurement system, for example, when the ratio of T to C (T / C) is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, the target amino group-containing compound can be evaluated as being capable of suppressing the increase in background.

[0051] The assay kit of the present disclosure can be suitably used as, for example, an assay kit, test kit, or research kit for measuring pTau217. Furthermore, the assay kit of the present disclosure can be suitably used to carry out the assay method of the present disclosure described below.

[0052] <Method for measuring phosphorylated tau protein> In another aspect, the present disclosure provides a measurement method capable of suppressing an increase in background signal caused by albumin, particularly bovine serum albumin. The method for measuring phosphorylated tau protein of the present disclosure includes a measurement step of contacting a subject sample with a measurement reagent for phosphorylated tau protein in which threonine phosphorylated at position 217 is phosphorylated in the presence of albumin and an amino group-containing compound, and measuring phosphorylated tau protein in which threonine phosphorylated at position 217 is phosphorylated in the sample. The measurement method of the present disclosure can suppress an albumin-dependent increase in background signal. Therefore, the measurement method of the present disclosure can improve the S / N ratio, for example, in a measurement system containing albumin. Since the measurement method of the present disclosure can suppress an increase in background signal caused by albumin in the measurement of phosphorylated tau protein, it can also be said to be a method for suppressing an increase in background signal caused by albumin in the measurement of phosphorylated tau protein, for example.

[0053] In the measuring step, a subject sample is contacted with the measuring reagent (measurement reagent for pTau217) of the present disclosure in the presence of the albumin and the amino group-containing compound, and phosphorylated tau protein in the sample, in which threonine at position 217 is phosphorylated, is measured. In the measuring step, for example, by measuring the presence or absence of pTau217 detected with the measurement reagent for pTau217, the presence or absence of pTau217 in the sample can be qualitatively measured. In addition, in the measuring step, for example, by measuring the amount of pTau217 detected with the measurement reagent for pTau217, the amount of pTau217 in the sample can be quantitatively measured.

[0054] In the measuring step, the measurement of pTau217 can be carried out using a binding molecule for pTau217. In this case, the measurement of pTau217 may be carried out by an immunological technique. Examples of the immunological technique include direct competitive ELISA, indirect competitive ELISA, sandwich ELISA, direct competitive immunoassay, indirect competitive immunoassay, sandwich immunoassay, immunochromatography, spin immunoassay, and latex agglutination. Furthermore, when the pTau217 measurement reagent is a reagent containing 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.

[0055] As an example of the measurement step, a measurement example in which the pTau217 is measured using the first binding molecule and a measurement example in which the pTau217 is measured using the first binding molecule and the second binding molecule will be described.

[0056] When the pTau217 is measured using the first binding molecule, the measurement step includes, for example, a complex formation step of contacting a sample with a first binding molecule for pTau217 in the presence of the albumin and an amino group-containing compound to form a first complex between pTau217 in the sample and the first binding molecule, and a complex measurement step of measuring pTau217 in the sample by measuring the first complex.

[0057] In the complex formation step, the contact can be performed, for example, by mixing the subject sample with the first binding molecule for pTau217. The contact is preferably performed in a liquid system containing water, physiological saline, the buffer solution, or the like.

[0058] In the complex formation step, the albumin concentration is, for example, 0.01 to 10 (w / v)%, 0.1 to 5 (w / v)%, or 0.5 to 3 (w / v)%. The albumin concentration can also be referred to as the concentration of a liquid system (reaction solution) containing the sample and the first binding molecule for pTau217.

[0059] In the complex formation step, the concentration of the amino group-containing compound is, for example, 1 to 200 mmol / L, 3 to 150 mmol / L, or 3.85 to 141.02 mmol / L, and is preferably 27.78 to 83.33 mmol / L, more preferably 28.85 to 76.92 mmol / L, since background can be effectively suppressed. The concentration of the amino group-containing compound can also be referred to as the concentration of a liquid system (reaction solution) containing the sample and a first binding molecule for pTau217.

[0060] When the amino group-containing compound contains Tris and the amino acid, the concentrations of Tris and the amino acid are, for example, as follows: Tris concentration: 19.23 to 83.33 mmol / L, 27.78 to 83.33 mmol / L Amino acid concentration: 3.85 to 57.69 mmol / L, preferably 9.62 to 57.69 mmol / L

[0061] In the complex formation step, the contact conditions (e.g., temperature, time, pH) between the sample and the first binding molecule are not particularly limited as long as they allow the first complex to be formed. 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 complex formation 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 measurement step, the albumin and the amino group-containing compound may be present when the sample is contacted with the pTau217 assay reagent (e.g., the first binding molecule and / or the second binding molecule), and the order of contacting is not particularly limited. In the measurement step, for example, the albumin and the amino group-containing compound may be contacted with the sample, and then the sample may be contacted with the pTau217 assay reagent (e.g., a solution containing the first binding molecule and / or the second binding molecule), or the albumin and the amino group-containing compound may be contacted with the pTau217 assay reagent (e.g., a solution containing the first binding molecule and / or the second binding molecule) and then the sample may be contacted, or the albumin and the amino group-containing compound may be contacted with the sample, and the pTau217 assay reagent (e.g., a solution containing the first binding molecule and / or the second binding molecule) may be contacted simultaneously. In the measurement step, the first binding molecule and / or the second binding molecule may be diluted with a first diluent and / or a second diluent containing the albumin and the amino group-containing compound, and the resulting diluted solution of the first binding molecule and / or the second binding molecule may be brought into contact with the sample.

[0063] The complex measuring step is, for example, a step of measuring a first complex of pTau217 and the first binding molecule in the sample, i.e., a step of measuring the binding between the pTau217 and the first binding molecule. In the complex measuring step, by detecting the presence or absence of binding between the two, for example, the presence or absence of pTau217 in the sample can be analyzed (qualitatively), and by detecting the degree of binding between the two (amount of binding), for example, the amount of pTau217 in the sample can be analyzed (quantitatively).

[0064] The method for measuring the binding between the pTau217 and the first binding molecule is not particularly limited, and for example, a conventionally known method for measuring binding between substances can be used, specifically, SPR, fluorescence polarization, etc. Furthermore, when the first binding molecule has the label, the binding between the pTau217 and the first binding molecule may be measured by directly or indirectly detecting the label in the first complex in the complex measurement step. The method for detecting the label can be appropriately determined depending on, for example, the type of the label.

[0065] Furthermore, when the pTau217 is measured using the first binding molecule and the second binding molecule, the measuring step includes a first complex formation step of contacting the sample with a first binding molecule for pTau217 to form a first complex between pTau217 in the sample and the first binding molecule, a second complex formation step of contacting the first complex with a second binding molecule for pTau217 to form a second complex between the first complex and the second binding molecule, and a complex measurement step of measuring pTau217 in the sample by measuring the second complex. When the measuring step includes the first complex formation step and the second complex formation step, the first complex formation step and / or the second complex formation step are preferably performed in the presence of albumin and the amino group-containing compound, since this can more effectively suppress an increase in background. When measuring pTau217 using the first binding molecule and the second binding molecule, it is preferable that the first binding molecule is supported on the carrier before, during, or after the formation of the first complex. When the first binding molecule is supported on the carrier after the formation of the first complex, the measurement method may, for example, involve supporting the first binding molecule on the carrier in the second complex formation step described below. When the first binding molecule is supported on the carrier during or after the formation of the first complex, the first binding molecule may, for example, be indirectly immobilized on the carrier. 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.

[0066] The first complex formation step can be carried out in the same manner as the complex formation step described above. When the first complex formation step is carried out in the presence of the albumin and the amino group-containing compound, the concentrations of the albumin and the amino group-containing compound can be determined in the same manner as the complex formation step described above.

[0067] In the measuring step, since the measurement accuracy of pTau217 can be improved after the first complex formation step, it is preferable to separate the first complex from other components, and optionally, it is more preferable to further wash the separated first complex with the washing solution. The separation of the first complex can be carried out, for example, by solid-liquid separation. When the first binding molecule is supported on a carrier, the 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 is supported on magnetic particles, the 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.

[0068] Next, in the second complex formation step, the first complex is contacted with the second binding molecule to form the second complex. The contact between the first complex and the second binding molecule is preferably carried out in a liquid system containing, for example, water, physiological saline, or the buffer solution. When the second complex formation step is carried out in the presence of the albumin and the amino group-containing compound, the concentrations of the albumin and the amino group-containing compound can be determined as described above for the complex formation step.

[0069] In the second complex formation step, the contact conditions between the first complex and the second binding molecule are not particularly limited as long as the conditions allow the formation of the second complex. Specific examples of the temperature for the contact include 4 to 42° C. and 18 to 40° C. The time for the contact includes 1 minute to 12 hours, 3 minutes to 120 minutes, and 5 minutes to 60 minutes.

[0070] In the measuring step, after the second complex formation step, it is preferable to separate the second complex because this can improve the measurement accuracy of the pTau217, and it is more preferable to optionally further wash the separated second complex with the washing solution. The separation of the second complex can be performed, for example, in the same manner as the separation of the first complex.

[0071] The complex measurement step is, for example, a step of measuring a second complex between the first complex and the second binding molecule, i.e., a step of measuring the binding between the first complex and the second binding molecule. In the complex measurement step, by detecting the presence or absence of binding between the two, for example, the presence or absence of pTau217 in the sample can be analyzed (qualitatively), and by detecting the degree of binding between the two (binding amount), for example, the amount of pTau217 in the sample can be analyzed (quantitatively). The method for measuring the binding between the first complex and the second binding molecule can be performed, for example, in the same manner as the method for measuring the binding between pTau217 and the first binding molecule. When the second binding molecule has a label, the complex measurement step may measure the binding between the first complex and the second binding molecule by directly or indirectly detecting the label in the second complex. The method for detecting the label can be determined appropriately depending on, for example, the type of label.

[0072] In the complex measurement step, for example, a second complex between the first complex and the second binding molecule may be separated, the separated second complex is washed with the washing solution, and then a label is released from the second complex and the released label is detected, thereby measuring the binding between the first complex and the second binding molecule.

[0073] In the measurement method of the present disclosure, the first complex formation step and the second complex formation step may be performed simultaneously. When the first complex formation step and the second complex formation step are performed simultaneously, they can be performed in the same manner as the complex formation step. When the first complex formation step and the second complex formation step are performed simultaneously, the measurement step may involve forming a complex (corresponding to the second complex) between pTau217, the first binding molecule, and the second binding molecule in the sample in the same reaction system. In this case, the measurement step may include, for example, a complex formation step in which the target sample is contacted with the first binding molecule and the second binding molecule in the presence of albumin and the amino group-containing compound to form a complex (corresponding to the second complex) between pTau217, the first binding molecule, and the second binding molecule in the sample, and a complex measurement (detection) step in which the complex is measured (detected).

[0074] In this way, the measurement method of the present disclosure can measure pTau217.

[0075] The measurement method of the present disclosure may, for example, optionally analyze the amount of pTau217 in the measurement step. In this case, the measurement step may, for example, determine in advance a correlation between the amount of pTau217 present and a count value (e.g., luminescence amount) obtained by measuring pTau217, and analyze the amount of pTau217 in the sample from the count value based on the correlation. Therefore, the measurement method of the present disclosure may also be referred to as, for example, an analysis method.

[0076] According to the measurement method of the present disclosure, an increase in background caused by albumin can be suppressed. Therefore, the measurement method of the present disclosure can improve the S / N ratio, for example, in the measurement of phosphorylated tau protein. Furthermore, it is known that the content of pTau217 in the biological sample is generally very small. The measurement method of the present disclosure can, for example, improve the S / N ratio, and therefore can also suitably measure pTau217 in the biological sample.

[0077] 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."

[0078] Reference Example 1 A measurement reagent was prepared as follows, and tau 217 in which threonine at position 217 was phosphorylated (phosphorylated tau 217, pTau217) was measured.

[0079] (1) Preparation of Immobilized Particle Solution: Magnetic particles immobilized with mouse anti-phosphorylated tau 217 antibody (pTau217), which specifically binds to phosphorylated tau 217, were diluted with a particle diluent (buffer, 150 mM NaCl, 1 mM EDTA 2Na, 0.1% ProClin 300, 2.0% BSA) to prepare an immobilized particle solution. The magnetic particles immobilized with mouse anti-phosphorylated tau 217 antibody were those included in Lumipulse® G pTau217 Immunoreaction Cartridges (manufactured by Fujirebio Europe).

[0080] (2) 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, purified by gel filtration, and F(ab') ２ The resulting fragments were then reduced and desalted, mixed with maleimide-conjugated alkaline phosphatase (ALP), and subjected to a coupling reaction. After quenching, the resulting fragments were desalted and purified by gel filtration to obtain the ALP-labeled antibody. The resulting fragments were mixed with a labeled antibody diluent (50 mM MOPS buffer, 1 mM MgCl2, 0.3 mM ZnCl2, 2.0% BSA, 150 mM NaCl, pH 6.8) to obtain an ALP-labeled antibody solution containing antibody A and antibody B.

[0081] (3) 100 μl of the phosphorylated tau 217 measurement sample or calibrator, 150 μl of the solid-phase particle solution, and 20 μl of the measurement reaction solution were dispensed into a reaction vessel, stirred, and the reaction solution was prepared and then incubated at 37°C for 10 minutes. Bound / Free (B / F) separation and washing were then performed. The measurement reaction solution was that included with the Lumipulse G pTau217 Immunoreaction Cartridges. Furthermore, Lumipulse washing solution (Fujirebio) was used for the washing. After the washing, 150 μl of the ALP-labeled antibody solution prepared above was dispensed into the reaction vessel, stirred, and then incubated at 37°C for 10 minutes. B / F separation and washing were then performed. After the 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. After stirring, the mixture was incubated at 37°C for 5 minutes, and the luminescence intensity was measured using a luminometer. Actual measurements were performed using a fully automated chemiluminescent enzyme immunoassay system, "Lumipulse G1200" (Fujirebio). Where necessary, the measured values ​​of phosphorylated tau 217 for each sample were calculated from the count values ​​(luminescence intensity) of each sample based on a calibration curve created using the count values ​​(luminescence intensity) of the calibrator. The concentration of BSA in the reaction solution was 1.11 (w / v)%. The concentration of BSA during the reaction with the labeled antibody solution was 2 (w / v)%.

[0082] Example 1 It was found that differences in BSA lots cause an increase in background in the measurement of phosphorylated tau 217, and that the increase in background can be suppressed by using a Tris buffer and adding glycine.

[0083] The present inventors measured standard phosphorylated tau 217 using the measurement method described in Reference Example 1 (3) above and found that the background level increased with each production lot. After investigating the cause, the present inventors came up with the idea that the background level might be increased due to differences in BSA lots. Therefore, they investigated whether the background level increased due to differences in BSA lots and investigated buffers and additives that suppressed the background level increase.

[0084] (1) Inhibition of phosphorylated tau 217 measurement due to differences in BSA lots. The luminescence intensity of phosphorylated tau 217 calibration samples (CAL1-5: 0.00, 0.25, 1.00, 5.00, or 10.00 pg / ml: standard phosphorylated tau 217 (plasma) solution (Fujirebio Europe) was measured using BSA lots No. 1 to No. 3 (Proliant Biologicals). The measurement method was based on the method described in Reference Example 1 (3) above. MOPS 50 mM (pH 7.2) was used as the buffer for the particle dilution solution. The S / N ratio (CAL2 / CAL1) was calculated using the count average (hereinafter referred to as "luminescence intensity") of CAL2 (0.25 pg / ml) and the luminescence intensity of CAL1 (0.00 pg / ml). The measurement results and S / N ratios are shown in Table 1 below.

[0085]

[0086] Table 1 shows the luminescence intensity and S / N ratio of each sample when each lot of BSA was used. As shown in Table 1, it was found that differences in the background and S / N ratio occurred depending on the lot of BSA.

[0087] (2) Investigation of buffers that suppress measurement inhibition. To suppress background increases due to differences in BSA lots, buffers were investigated. Measurement of phosphorylated tau 217 calibration samples (CAL 1 and CAL 2) was performed in the same manner as in Example 1(1), except that 50 mM MOPS (pH 7.2), 50 mM Tris (pH 7.2), or 150 mM Tris (pH 7.2) was added as the buffer for the particle dilution solution. The S / N ratio (CAL 2 / CAL 1) was calculated using the luminescence intensity of CAL 2 (0.25 pg / ml) and the luminescence intensity of CAL 1 (0.00 pg / ml). The ratio of the luminescence intensity of each buffer was calculated based on the luminescence intensity of 50 mM MOPS. The luminescence intensity, S / N ratio, and luminescence intensity ratio (relative to MOPS 50 mM (CAL 1 (0.00 pg / mL)) (S / C (%))) are shown in Table 2 below.

[0088]

[0089] Table 2 shows the luminescence intensity, S / N ratio, and luminescence intensity ratio for each sample when each buffer was used. As shown in Table 2, the 50 mM Tris buffer had a lower luminescence intensity and a higher S / N ratio for CAL 1 than the 50 mM MOPS buffer. This indicates that the 50 mM Tris buffer can suppress background increases compared to the 50 mM MOPS buffer. Furthermore, the 150 mM Tris buffer had a lower luminescence intensity and a higher S / N ratio for CAL 1 than the 50 mM Tris buffer. This indicates that the Tris buffer can suppress background increases caused by BSA in a Tris concentration-dependent manner.

[0090] (3) Investigation of Additives That Suppress Measurement Inhibition Next, we investigated whether the addition of glycine to the particle dilution or the labeled antibody dilution suppresses background increases. 50 mM Tris buffer was used as the buffer for the particle dilution. Measurements of phosphorylated tau 217 calibration samples (CAL 1, CAL 2) were performed in the same manner as in Example 1(1), except that glycine was added or not added to the particle dilution or labeled antibody dilution. The S / N ratio (CAL 2 / CAL 1) was calculated using the luminescence intensity of CAL 2 (0.25 pg / ml) and the luminescence intensity of CAL 1 (0.00 pg / ml). Furthermore, to detect the effect of suppressing background increases from the luminescence intensity, the ratio of the luminescence intensity of CAL 1 with glycine (S) to the luminescence intensity of CAL 1 without glycine (C) was calculated (S / C × 100 (%)). The luminescence intensity, S / N ratio, and luminescence intensity ratio are shown in Table 3 below.

[0091]

[0092] Table 3 shows the luminescence intensity, S / N ratio, and luminescence intensity ratio of each sample after the addition of glycine. As shown in Table 3, compared to condition 1 (no glycine added), the samples in which glycine was added to the particle dilution or the labeled antibody dilution to a concentration of 50 mM had lower CAL 1 measurements and higher S / N ratios. Furthermore, compared to condition 1 (no glycine added), conditions 2 and 3 (in which glycine was added to the particle dilution or the labeled antibody dilution to a concentration of 50 mM) reduced the background increase by approximately 8%. This indicates that the addition of glycine to the particle dilution or the labeled antibody dilution to a glycine concentration of 50 mM can suppress the background increase caused by BSA, compared to condition 1 (no glycine added). Furthermore, condition 4 (in which glycine was added to the particle dilution or the labeled antibody dilution to a concentration of 50 mM) reduced the background increase by approximately 20% compared to condition 1 (no glycine added).

[0093] These results demonstrate that the background increases depending on the BSA lot, and that the increase in background can be suppressed by using a Tris buffer and adding glycine. Furthermore, the addition of glycine synergistically suppresses background in both the primary reaction with the diluted particle solution and the secondary reaction with the diluted labeled antibody solution.

[0094] Example 2 It was confirmed that the increase in background in the measurement of phosphorylated tau 217 can be suppressed by adding glycine at various concentrations to a particle dilution solution.

[0095] To suppress background increase, the concentration of glycine added to the particle dilution was investigated. Measurements of phosphorylated tau 217 calibration samples (CAL 1 to CAL 3) were performed in the same manner as in Example 1(1), except that 50 mM Tris buffer was used as the buffer for the particle dilution, with or without glycine added; 50 mM glycine was added to the labeled antibody dilution; and sample volumes of 50 μl, 50 μl of solid-phase particle solution, 30 μl of measurement reaction solution, and 50 μl of labeled antibody solution were used. The S / N ratio (CAL 2 / CAL 1) was calculated using the luminescence intensity of CAL 2 (0.25 pg / ml) and the luminescence intensity of CAL 1 (0.00 pg / ml). Furthermore, to detect the effect of suppressing background increase from the obtained measurements, the ratio (S) of the luminescence intensity of the particle dilution with glycine added to the luminescence intensity of the particle dilution without glycine added (C) was calculated (S / C × 100 (%)). The luminescence intensity, S / N ratio, and luminescence intensity ratio are shown in Table 4 below.

[0096]

[0097] Table 4 shows the luminescence intensity, S / N ratio, and luminescence intensity ratio of each sample after addition of glycine. As shown in Table 4, samples containing glycine added to the particle dilution solution at a concentration of 10 to 150 mM had lower background and higher S / N ratios than samples containing no glycine. This indicates that, compared to samples containing no glycine, the addition of glycine to the particle dilution solution (Tris concentration in the reaction solution: 19.23 mM) to achieve a glycine concentration of 10 to 150 mM (reaction solution: approximately 3.85 to 57.69 mM) can suppress the increase in background intensity due to BSA. Furthermore, samples containing glycine added to the particle dilution solution at a concentration of approximately 25 to 150 mM (reaction solution: approximately 9.62 to 57.69 mM) had lower luminescence intensity of CAL 1 and higher S / N ratios, indicating that the increase in background intensity due to BSA can be further suppressed compared to samples containing other concentrations of glycine.

[0098] From the above, it was found that adding glycine at various concentrations to the particle dilution solution can suppress an increase in background in the measurement of phosphorylated tau 217. In particular, it was found that adding glycine to the particle dilution solution at 25 to 150 mM, i.e., so that the glycine concentration in the reaction solution becomes 9.62 to 57.69 mM, is highly effective in suppressing an increase in background caused by BSA.

[0099] Example 3 It was confirmed that in the measurement of phosphorylated tau 217 in human plasma samples, an increase in background caused by BSA could be suppressed even when a glycine-containing Tris buffer solution was used.

[0100] The buffer and additives used in the particle dilution solution were examined to determine whether they could suppress background increases in the measurement of phosphorylated tau 217 in human plasma samples. Measurement of phosphorylated tau 217 in calibration samples (CAL 1-5) and human plasma samples was carried out in the same manner as in Example 1(1), except that either 50 mM MOPS buffer or 50 mM Tris buffer containing 50 mM glycine was used as the buffer for the particle dilution solution. The S / N ratio (CAL 2 / CAL 1) was calculated using the luminescence intensity at CAL 2 (0.25 pg / ml) and the luminescence intensity at CAL 1 (0.00 pg / ml). The concentration (measured value) of phosphorylated tau 217 was calculated from the count value (luminescence intensity) of each plasma sample based on a calibration curve constructed using the count values ​​(luminescence intensity) of each calibrator sample. Furthermore, to detect the effect of suppressing background increase, the ratio (S / C) of the luminescence intensity (S) of each plasma sample to the luminescence intensity (C) of CAL 1 of each particle dilution is shown in Table 5 below.

[0101]

[0102] Table 5 shows the luminescence intensity, measured value, S / N ratio, and S / C ratio of each sample with the addition of glycine. As shown in Table 5, the average measured values ​​of all plasma samples did not differ significantly depending on the buffer. In contrast, compared to 50 mM MOPS buffer, 50 mM Tris buffer containing 50 mM glycine had a lower luminescence intensity and a higher S / N ratio for CAL 1. Furthermore, compared to 50 mM MOPS buffer, 50 mM Tris buffer containing 50 mM glycine had a higher ratio of luminescence intensity of each plasma sample to CAL 1 (S / C). In other words, it was found that even in plasma samples, the increase in background intensity due to BSA can be suppressed by using glycine-containing Tris buffer as the particle dilution solution.

[0103] Example 4 It was confirmed that the increase in background in the measurement of phosphorylated tau 217 can be suppressed by adding an amino acid other than glycine to the particle dilution and the labeled antibody dilution.

[0104] Additives other than glycine were added to the particle dilution and the labeled antibody dilution to investigate whether they could suppress background increases. Measurement of phosphorylated tau 217 calibration samples (CAL 1-3) was performed in the same manner as in Example 2, except that 50 mM Tris buffer was used as the buffer for the particle dilution, 50 mM Tris buffer was used instead of 50 mM MOPS buffer as the buffer for the labeled antibody dilution, and glycine, alanine, serine, histidine, or tryptophan was added to these buffers. The S / N ratio (CAL 2 / CAL 1) was calculated using the luminescence intensity of CAL 2 (0.25 pg / ml) and the luminescence intensity of CAL 1 (0.00 pg / ml). The luminescence intensity and S / N ratio are shown in Table 6 below.

[0105]

[0106] Table 6 shows the luminescence intensity and S / N ratio of each sample with the addition of each amino acid. As shown in Table 6, the samples to which alanine, serine, histidine, or tryptophan was added to the particle dilution and the labeled antibody dilution achieved CAL 1 luminescence intensity and S / N ratios equivalent to those obtained with the addition of glycine. This indicates that adding amino acids other than glycine, such as alanine, serine, histidine, or tryptophan, to the particle dilution and the labeled antibody dilution can suppress the increase in background signal caused by BSA, similar to glycine. Furthermore, it was found that, among amino acids, glycine specifically reduces the increase in background signal caused by BSA and most effectively improves the S / N ratio.

[0107] 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.

[0108] This application claims priority based on Japanese Patent Application No. 2024-124420, filed on July 31, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0109] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendixes. <Method for measuring phosphorylated tau protein> (Appendix 1) A method for measuring phosphorylated tau protein, comprising a measurement step of contacting a subject sample with a measurement reagent for measuring phosphorylated tau protein (pTau217) in the presence of albumin and an amino group-containing compound, and measuring pTau217 in the sample. (Appendix 2) The measurement method according to Appendix 1, wherein the amino group-containing compound comprises tris(hydroxymethyl)aminomethane (Tris) and / or an amino acid. (Appendix 3) The measurement method according to Appendix 1 or 2, wherein the amino acid comprises glycine. (Appendix 4) The measurement method according to any of Appendixes 1 to 3, wherein the concentration of the amino group-containing compound is 3.85 to 141.02 mmol / L. (Supplementary Note 5) The measurement method according to Supplementary Note 1, wherein the amino group-containing compound comprises tris(hydroxymethyl)aminomethane (Tris) and glycine. (Supplementary Note 6) The measurement method according to Supplementary Note 5, wherein the concentration of Tris is 19.23 to 83.33 mmol / L, and the concentration of glycine is 3.85 to 57.69 mmol / L. (Supplementary Note 7) The measurement method according to any one of Supplementary Notes 1 to 6, wherein the measuring step comprises: a first complex formation step of contacting the subject sample with a first binding molecule that binds to phosphorylated tau protein to form a first complex between the phosphorylated tau protein in the sample and the first binding molecule; a second complex formation step of contacting the first complex with a second binding molecule that binds to the phosphorylated tau protein to form a second complex between the first complex and the second binding molecule; and a detection step of detecting the second complex, wherein the first binding molecule or the second binding molecule specifically binds to pTau217, and the first complex formation step and / or the second complex formation step are performed in the presence of the albumin and the amino group-containing compound.(Supplementary Note 8) The measurement method according to any one of Supplementary Notes 1 to 6, wherein the measurement step comprises: a complex formation step of contacting the subject sample with a first binding molecule that binds to phosphorylated tau protein and a second binding molecule that binds to the phosphorylated tau protein to form a complex between the phosphorylated tau protein, the first binding molecule, and the second binding molecule; and a detection step of detecting the complex, wherein the first binding molecule or the second binding molecule specifically binds to pTau217, and the complex formation step is performed in the presence of the albumin and the amino group-containing compound. (Supplementary Note 9) The measurement method according to Supplementary Note 7 or 8, wherein the first binding molecule and / or the second binding molecule are supported on a carrier. (Supplementary Note 10) The measurement method according to Supplementary Note 9, wherein the carrier is a magnetic particle. (Supplementary Note 11) The measurement method according to any one of Supplementary Notes 7 to 10, wherein the first binding molecule and / or the second binding molecule has a label. (Appendix 12) The measurement method according to Appendices 11, wherein the detection step detects a label in the second complex. (Appendix 13) The measurement method according to any of Appendices 1 to 12, wherein the albumin is bovine serum albumin. (Appendix 14) The measurement method according to any of Appendices 1 to 13, wherein the measurement step suppresses an albumin-dependent background increase during the measurement by causing the albumin to coexist with an amino group-containing compound. <Kit for measuring phosphorylated tau protein> (Appendix 15) A kit for measuring phosphorylated tau protein, comprising: an amino group-containing compound; albumin; and a reagent for measuring phosphorylated tau protein (pTau217) in which threonine at position 217 is phosphorylated. (Supplementary Note 16) The measurement kit according to Supplementary Note 15, wherein the measurement reagent comprises: a first binding molecule that binds to the phosphorylated tau protein; and a second binding molecule that binds to the phosphorylated tau protein, wherein the first binding molecule or the second binding molecule is a binding molecule that specifically binds to pTau217. (Supplementary Note 17) The measurement kit according to Supplementary Note 16, wherein the first binding molecule and / or the second binding molecule comprises the albumin and the amino group-containing compound.(Appendix 18) The measurement kit according to Appendix 16, comprising a dilution of the first binding molecule and / or a dilution of the second binding molecule, wherein the dilution of the first binding molecule and / or the dilution of the second binding molecule contains the albumin and the amino group-containing compound. (Appendix 19) The measurement kit according to any of Appendixes 16 to 18, wherein the first binding molecule and / or the second binding molecule is supported on a carrier. (Appendix 20) The measurement kit according to Appendix 19, wherein the carrier is a magnetic particle. (Appendix 21) The measurement kit according to any of Appendixes 16 to 20, wherein the first binding molecule and / or the second binding molecule has a label. (Appendix 22) The measurement kit according to any of Appendixes 16 to 21, wherein the first binding molecule and / or the second binding molecule is an antibody. (Appendix 23) The measurement kit according to any one of Appendices 15 to 22, comprising a sample dilution solution, wherein the sample dilution solution contains the albumin and the amino group-containing compound. (Appendix 24) The measurement kit according to any one of Appendices 15 to 23, wherein the amino group-containing compound contains tris(hydroxymethyl)aminomethane and / or an amino acid. (Appendix 25) The measurement kit according to Appendices 24, wherein the amino acid contains glycine. (Appendix 26) The measurement kit according to any one of Appendices 15 to 23, wherein the amino group-containing compound contains tris(hydroxymethyl)aminomethane and glycine. (Appendix 27) The measurement kit according to any one of Appendices 15 to 26, wherein the albumin is bovine serum albumin. (Appendix 28) The measurement kit according to any one of Appendices 15 to 27, wherein the albumin-dependent background increase is suppressed by the amino group-containing compound. (Appendix 29) A measuring kit according to any one of Appendices 15 to 28, for use in the measuring method according to any one of Appendices 1 to 14.

[0110] As described above, the present disclosure provides a measurement method that can suppress an increase in background level due to albumin, particularly bovine serum albumin, and a measurement 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 method for measuring phosphorylated tau protein, comprising a measurement step of contacting a subject sample with a measurement reagent for phosphorylated tau protein (pTau217) in which threonine at position 217 is phosphorylated in the presence of albumin and an amino group-containing compound, and measuring pTau217 in the sample.

2. The measurement method according to claim 1, wherein the amino group-containing compound includes tris(hydroxymethyl)aminomethane (Tris) and / or an amino acid.

3. The measurement method according to claim 1 or 2, wherein the amino acid includes glycine.

4. The measurement method according to any one of claims 1 to 3, wherein the concentration of the amino group-containing compound is 3.85 to 141.02 mmol / L.

5. The measurement method according to claim 1, wherein the amino group-containing compound includes tris(hydroxymethyl)aminomethane (Tris) and glycine.

6. The measurement method according to claim 5, wherein the concentration of Tris is 19.23 to 83.33 mmol / L, and the concentration of glycine is 3.85 to 57.69 mmol / L.

7. A measurement method according to any one of claims 1 to 5, wherein the albumin is bovine serum albumin.

8. A measurement method according to any one of claims 1 to 7, wherein in the measurement step, the albumin and an amino group-containing compound are allowed to coexist, thereby suppressing an albumin-dependent background increase during the measurement.

9. A kit for measuring phosphorylated tau protein, comprising: an amino group-containing compound; albumin; and a reagent for measuring phosphorylated tau protein (pTau217) in which threonine at position 217 is phosphorylated.

10. The assay kit according to claim 9, wherein the assay reagent comprises: a first binding molecule that binds to the phosphorylated tau protein; and a second binding molecule that binds to the phosphorylated tau protein, and the first binding molecule or the second binding molecule is a binding molecule that specifically binds to pTau217.

11. The assay kit according to claim 9 or 10, wherein the amino group-containing compound comprises tris(hydroxymethyl)aminomethane and / or an amino acid.

12. The assay kit according to claim 11, wherein the amino acid includes glycine.

13. The measuring kit according to claim 9 or 10, wherein the albumin-dependent background increase is suppressed by the amino group-containing compound.

14. A measuring kit according to any one of claims 9 to 13 for use in the measuring method according to any one of claims 1 to 8.

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