Method for detecting tau protein using a blood sample

Abstract

This method comprises using two kinds of antibodies specifically binding to a tau protein or a phosphorylated tau protein. One of these antibodies is a first antibody that is used in a state of being immobilized on a support or labeled with a molecule capable of binding to the support, and the other is a second antibody that is labeled without being immobilized on the support. When the tau protein is divided into an N-terminal domain, a C-terminal domain and an intermediate domain positioned therebetween, epitopes respectively recognized by the first antibody and the second antibody are an amino acid sequence contained in the intermediate domain or an amino acid sequence contained in the N-terminal domain. One of these two antibodies is first subjected to an antigen-antibody reaction with a blood sample and subsequently the other is subjected to an antigen-antibody reaction.

Description

[Technical Field]

[0001] The present invention relates to a tau protein detection method using a blood sample as a specimen, and in particular to a tau protein detection method that can effectively detect tau protein contained in exosomes in a blood sample. [Background technology]

[0002] Tau protein is a protein that is mainly expressed in neurons and is known as a microtubule-associated protein (MAP) that promotes microtubule polymerization and stabilizes microtubules.

[0003] Tau protein is known as a phosphorylated protein in which multiple serine or threonine residues in its amino acid sequence are phosphorylated, and abnormalities in this phosphorylation are thought to be involved in neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies. Therefore, the use of tau protein and phosphorylated tau protein as biomarkers for neurodegenerative diseases has been investigated.

[0004] A specific method for detecting tau protein or phosphorylated tau protein is known, for example, as the method disclosed in Patent Document 1. Patent Document 1 lists molecules other than tau protein and phosphorylated tau protein as biomarkers, and lists biological samples such as blood (whole blood), serum, plasma, urine, interstitial fluid, ascites, cervical swab, tears, saliva, cheek swab, skin, brain tissue, and cerebrospinal fluid as specimens for detecting these biomarkers.

[0005] Furthermore, in Patent Document 1, vesicles such as exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, and ectosomes are isolated from these biological samples or these vesicles in the biological samples are concentrated, and one or more biomarkers are detected from such vesicles. If the biomarker is phosphorylated tau protein, the phosphorylated tau protein is detected using a composition containing an antibody.

[0006] Incidentally, as described in Patent Document 1, a biological sample that is particularly easy to use as a specimen can be a blood sample. However, because the content of tau protein in blood has previously been considered to be a minute amount, on the order of a few to a few tens of picograms per mL, highly sensitive detection systems for tau protein have been investigated. However, it has recently become clear that a portion of tau protein in blood exists in exosomes. Therefore, by extracting and using exosomes from a biological sample as described in Patent Document 1, it has become possible to detect tau protein at the level of several hundred picograms per mL of biological sample.

[0007] Meanwhile, methods for detecting tau protein directly from blood samples without extracting exosomes have also been investigated. For example, Patent Document 2 discloses a method in which, in the presence of non-capture beads, an immune complex is formed on capture beads between phosphorylated tau protein in a biological sample, a capture antibody, and a detection antibody, and a signal derived from the immune complex is detected. In Patent Document 2, the epitopes of the capture antibody and the detection antibody are different, and non-capture beads that do not form immune complexes are coexisted at a ratio of 1.5 to 1 capture bead.

[0008] According to Patent Document 2, by forming an immune complex by allowing non-capture beads to coexist in an amount 1.5 times or more greater than that of capture beads, phosphorylated tau protein can be measured (detected) with a sensitivity that allows Alzheimer's disease (AD) patients to be distinguished from control patients. However, there is no specific description of why the presence of non-capture beads enables phosphorylated tau protein to be detected (measured) well. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Special Publication No. 2016-535283 [Patent Document 2] Japanese Patent Application Publication No. 2019-027952 Summary of the Invention [Problem to be solved by the invention]

[0010] The inventors extracted and disrupted exosomes from blood samples, and confirmed the amount of exosomal protein by electrophoresis, revealing nanogram-level amounts of tau protein per mL. Therefore, it was thought that if tau protein contained in exosomes from blood samples could be detected with high reactivity, the accuracy of tau protein detection could be improved. However, conventional methods require the extraction of vesicles such as exosomes, making tau protein detection methods cumbersome.

[0011] Furthermore, it is anticipated that some of the tau protein in a blood sample may be lost during the process of extracting exosomes from the blood sample. For example, in Patent Document 1, drugs, antibodies, flow cytometry, or the like are used to isolate or concentrate vesicles such as exosomes from a biological sample, but depending on the conditions for isolation or concentration, some of the tau protein may be lost (a partial loss may occur), and this loss of tau protein may affect the detection results.

[0012] On the other hand, the loss of tau protein can be avoided by a method that uses a blood sample such as plasma directly without extracting exosomes, as in Patent Document 2. However, Patent Document 2 requires the use of a greater number of non-capture beads than the capture beads in addition to the capture beads for forming an immune complex. Therefore, as a method for detecting tau protein, the use of a large number of non-capture beads may make the detection process complicated.

[0013] Furthermore, in the examples of Patent Document 2, the proportion of non-capture beads is examined and the results of distinguishing between AD patients and control patients are shown, but the detection accuracy of tau protein, etc. is not specifically shown. Therefore, it is not clear how sensitively tau protein can be detected by the method disclosed in Patent Document 2.

[0014] The present invention has been made to solve such problems, and aims to provide a detection method that uses a blood sample as an analyte, avoids or reduces the complexity of the detection process, and is capable of detecting tau protein or phosphorylated tau protein with better sensitivity. [Means for solving the problem]

[0015] In order to solve the above-mentioned problems, the tau protein detection method of the present invention is a tau protein detection method that uses two types of antibodies that specifically bind to tau protein or phosphorylated tau protein to detect tau protein or phosphorylated tau protein in a specimen collected from a subject through an antigen-antibody reaction, wherein the specimen is a blood sample, one of the two types of antibodies is a first antibody that is immobilized on a carrier or labeled with a molecule that can bind to the carrier, and the other is a second antibody that is not immobilized on the carrier and is labeled, when the tau protein is divided into an N-terminal region, a C-terminal region, and an intermediate region located therebetween, the epitopes recognized by the first antibody and the second antibody are amino acid sequences contained in the intermediate region or amino acid sequences contained in the N-terminal region, and one of the two types of antibodies is first subjected to an antigen-antibody reaction with the blood sample, and then the other is subjected to an antigen-antibody reaction with the blood sample.

[0016] According to the above configuration, tau protein or phosphorylated tau protein in a sample is detected by a sandwich method that combines a substantially immobilized first antibody with a non-immobilized, labeled second antibody. In this case, antibodies having an epitope in the intermediate region or N-terminal region of tau protein are used as the first and second antibodies, rather than antibodies having an epitope in the C-terminal region of tau protein. Furthermore, a non-immobilized support on which no antibody is immobilized (a non-capture support on which no antibody is immobilized as a capture molecule) is not used in addition to a solid-phase support on which the first antibody is immobilized (a capture support on which an antibody serving as a capture molecule is immobilized).

[0017] This method enables the effective detection of tau protein or phosphorylated tau protein contained in exosomes from blood samples without pretreatment such as exosome extraction. This effectively avoids the possibility of tau protein loss during the exosome extraction process, allowing for more sensitive detection of tau protein (or phosphorylated tau protein). Furthermore, not only does it eliminate the need for exosome extraction, but it also eliminates the need to prepare a non-immobilized support or adjust the ratio of the non-immobilized support to the immobilized support. This effectively reduces or avoids the complexity of tau protein detection. Furthermore, since antibodies that bind to the C-terminal region do not need to be used as the first and second antibodies, the flexibility in selecting the two types of antibodies is improved.

[0018] In the tau protein detection method having the above configuration, when tau 441 protein, which has the longest amino acid sequence of 441 residues among the tau protein isoforms, is used as a standard, the N-terminal region may be a region consisting of the amino acid sequence from the 1st to the 44th amino acids of the tau 441 protein, and the intermediate region may be a region consisting of the amino acid sequence from the 103rd to the 371st amino acids of the tau 441 protein.

[0019] Furthermore, in the tau protein detection method having the above configuration, when the amino acid sequence that serves as the epitope of the antibody is contained in the intermediate region, the epitope may be contained in a proline-rich region consisting of the amino acid sequence from the 149th to the 244th amino acids in the intermediate region.

[0020] Furthermore, in the tau protein detection method having the above configuration, when the amino acid sequence that serves as the epitope of the antibody is contained in the intermediate region, the epitope may be contained in a proline-rich region consisting of the amino acid sequence from the 188th to the 244th amino acids in the intermediate region.

[0021] Furthermore, in the tau protein detection method having the above configuration, when tau 441 protein, which has the longest amino acid sequence of 441 residues among the tau protein isoforms, is used as a standard, the phosphorylated tau protein may be configured such that at least one of the amino acid residues at positions 46, 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, 238, 262, and 356 of the tau 441 protein is phosphorylated.

[0022] Furthermore, in the tau protein detection method having the above configuration, the phosphorylated tau protein that serves as the antigen for the first antibody may be configured such that at least one of the amino acid residues at positions 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, and 238 of the tau 441 protein is phosphorylated.

[0023] Furthermore, in the tau protein detection method having the above configuration, the carrier to which the first antibody is immobilized may be magnetic beads, resin beads, glass beads, a resin plate, a membrane, or a resin tube, and the molecule capable of binding to the carrier may be biotin or avidin.

[0024] Furthermore, in the tau protein detection method configured as above, the label on the second antibody may be an enzyme, a fluorescent dye, a fluorescent protein, or biotin.

[0025] Furthermore, in the tau protein detection method configured as above, the specimen may be a blood sample obtained from a subject suspected of having a neurodegenerative disease.

[0026] Furthermore, in the tau protein detection method configured as above, the neurodegenerative disease may be at least one of Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies.

[0027] The present invention may also include a method for identifying a neurodegenerative disease by detecting tau protein or phosphorylated tau protein in the blood sample using the tau protein detection method configured as described above.

[0028] The above and other objects, features, and advantages of the present invention will become apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings. [Effects of the Invention]

[0029] With the above-described configuration, the present invention has the effect of providing a detection method that uses a blood sample as a specimen, avoids or suppresses the complexity of the detection process, and is capable of detecting tau protein or phosphorylated tau protein with better sensitivity. [Brief explanation of the drawings]

[0030] [Figure 1] 1 is a schematic diagram illustrating the structure of tau protein, which is the detection target of the tau protein detection method according to the present invention. FIG. [Figure 2] 1 is a graph showing a comparison of the results of tau protein detection in Example 2 and Reference Example 1 in the tau protein detection method according to the present invention. [Figure 3] 1 is a graph showing a comparison of the results of tau protein detection in Example 3 and Reference Example 2 in the tau protein detection method according to the present invention. [Figure 4] 1 is a graph showing a comparison of the results of tau protein detection in Comparative Example 1 and Reference Example 3 in the tau protein detection method according to the present invention. [Figure 5] 1 is a graph showing a comparison of the detection results of phosphorylated tau protein in Example 8 and Reference Example 4 in the tau protein detection method according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] Representative embodiments of the present invention will be described in detail below. The tau protein detection method according to the present disclosure is a method for detecting tau protein or phosphorylated tau protein from a specimen collected from a subject through an antigen-antibody reaction using two types of antibodies (anti-tau antibodies or anti-phosphorylated tau antibodies) that specifically bind to tau protein or phosphorylated tau protein. A blood sample is used as the specimen, and two types of anti-tau antibodies or anti-phosphorylated tau antibodies are used in combination: a first antibody that is immobilized on a carrier or labeled with a molecule that can bind to the carrier (substantially immobilized), and a second antibody that is not immobilized on a carrier but is labeled (not immobilized). In this disclosure, for ease of explanation, anti-tau antibodies that specifically bind to tau protein and anti-phosphorylated tau antibodies that specifically bind to phosphorylated tau protein will be collectively abbreviated as "tau antibodies."

[0032] Here, when tau protein is divided into an N-terminal region, a C-terminal region, and an intermediate region located between them, the epitopes recognized by the first antibody and the second antibody, respectively, are amino acid sequences contained in the intermediate region or amino acid sequences contained in the N-terminal region. That is, the first antibody may be a tau antibody whose epitope is an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein, or a tau antibody whose epitope is an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein. Similarly, the second antibody may be a tau antibody whose epitope is an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein, or a tau antibody whose epitope is an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein.

[0033] In the tau protein detection method according to the present disclosure, one of the two types of tau antibodies may be subjected to an antigen-antibody reaction with a blood sample first, and then the other may be subjected to an antigen-antibody reaction with the blood sample. For example, a first reaction solution may be prepared by mixing a second antibody that has not been immobilized with the blood sample and causing an antigen-antibody reaction, and then the first reaction solution may be subjected to an antigen-antibody reaction with the immobilized first antibody. Alternatively, a first reaction solution may be prepared by causing an antigen-antibody reaction with a blood sample with a first antibody that has been immobilized, and then the first reaction solution may be subjected to an antigen-antibody reaction with the second antibody that has not been immobilized.

[0034] [Tau protein and phosphorylated tau protein] The tau protein or phosphorylated tau protein to be detected in the present disclosure is described in Patent Document 1 or Patent Document 2, and in Reference Document 1: Hasan AMM Almansoub, et. al., "Tau Abnormalities and the Potential Therapy in Alzheimer's Disease," Journal of Alzheimer's Disease 67 (2019) pp. 13-33, or Reference Document 2: WO 2013 / 180238, etc. Phosphorylated tau protein is tau protein in which some of the amino acids constituting the protein are phosphorylated.

[0035] As described in Reference 1 or Reference 2, six typical isoforms of tau protein are known. The MAPT gene, which encodes tau protein, has 16 exons, and exons 1 to 13 are expressed in the central nervous system. Of these, exons 2, 3, and 10 undergo alternative splicing, resulting in the six isoforms described above.

[0036] Specifically, alternative splicing of exon 2 or exon 3 of the MAPT gene at the N-terminus results in 0 to 2 repeats N (0N-2N), whereas alternative splicing of exon 10 of the MAPT gene results in 3 (1R-3R) or 4 (1R-4R) repeats R in the microtubule-binding region. As described in Reference 1, the six isoforms of tau protein, based on the differences in these repeats, can be expressed as 0N3R (352 amino acid residues), 1N3R (381 amino acid residues), 2N3R (410 amino acid residues), 0N4R (383 amino acid residues), 1N4R (412 amino acid residues), and 2N4R (441 amino acid residues) (although some isoforms, such as Reference 2, have reversed N and R). The tau protein to be detected in the present disclosure includes all six isoforms.

[0037] In the present disclosure, the ordinal numbers of amino acid sequences are defined based on 2N4R, an isoform in which alternative splicing does not occur in any of exons 2, 3, and 10, i.e., tau protein 2N4R, which has the longest amino acid sequence of 441 residues among the six isoforms. For ease of explanation, in this embodiment, tau protein 2N4R will be referred to as "tau 441 protein" based on the number of amino acid residues.

[0038] As described in Reference 1 and shown in Figure 1, the tau441 protein (2N4R) can be divided into four regions: the N-terminal region, the proline-rich domain (PRD), the microtubule-binding domain (MBD), and the C-terminal region. The N-terminal region consists of amino acids 1 to 148 (aa1-148), the proline-rich domain consists of amino acids 149 to 244 (aa149-244), the microtubule-binding domain consists of amino acids 245 to 372 (aa244-372), and the C-terminal region consists of amino acids 373 to 441 (aa373-441). In Figure 1, the two N-terminal repeats N are referred to as "N1" and "N2," respectively, and the four R repeats in the microtubule-binding domain are referred to as "R1," "R2," "R3," and "R4," respectively.

[0039] In the present disclosure, the amino acid sequence of a tau protein recognized by a tau antibody, i.e., the amino acid sequence serving as the epitope of a tau antibody, is contained at least in the intermediate region, and further in the N-terminal region in addition to the intermediate region. Here, the intermediate region of a tau protein according to the present disclosure may be any region located between the N-terminal region and the C-terminal region, and therefore, the intermediate region may be a region including a proline-rich region and a microtubule-binding region, i.e., a region consisting of amino acids 149 to 372 (aa149-372).

[0040] In the present disclosure, the N-terminal region is defined as a broad region extending up to just before the proline-rich region, but the narrow region from the N-terminus to just before the repeat sequence N can be defined as the "narrowly defined" N-terminal region. This is because tau protein isoforms include ON3R and ON4R, which do not contain the repeat sequence N. In other words, if the amino acid sequence recognized by a tau antibody is included in the "narrowly defined" N-terminal region, the ON3R and ON4R isoforms can also be successfully detected. Specifically, the "narrowly defined" N-terminal region is composed of the amino acid sequence from the 1st to the 44th amino acids (aa1-44).

[0041] If the N-terminal region is the "narrowly defined" region, then the intermediate region located between the N-terminal region and the C-terminal region can be defined as the region extending toward the N-terminus. In other words, the reference N-terminal region is composed of the amino acid sequence from 1 to 148 (aa1-148), while the "narrowly defined" N-terminal region is composed of the amino acid sequence from 1 to 44 (aa1-44), as mentioned above. Therefore, the "broadly defined" intermediate region can be expanded to the 45th amino acid residue on the N-terminus side.

[0042] However, as described above, since repeat sequence N may not be included due to alternative splicing, when using tau441 protein as a reference, the start point of the intermediate region can be set to the amino acid residue next to the end point of repeat sequence N. Since the end point of repeat sequence N is the 102nd amino acid residue of tau441 protein, the start point of the "broad" intermediate region can be set to the 103rd amino acid residue. Therefore, in this embodiment, the "broad" intermediate region preferably consists of the amino acid sequence from the 103rd to the 372nd amino acid residues (aa103-372).

[0043] Furthermore, the "broad" intermediate region and the "narrow" N-terminal region in the present disclosure can also be defined and expressed based on the repeat sequence N and the repeat sequence R rather than based on the ordinal numbers of the amino acid sequence of the Tau441 protein.

[0044] Specifically, for example, the end point of repeat sequence N, i.e., the amino acid sequence encoded by exon 2 or exon 3 of the MAPT gene, may be the start point of the "broad" intermediate region, or the position where the amino acid sequences encoded by exons 2 and 3 are deleted due to alternative splicing may be the start point of the "broad" intermediate region. Furthermore, the end point of the "broad" intermediate region may be the end point of repeat sequence R, i.e., the amino acid sequence encoded by exons 9 to 12 of the MAPT gene. Similarly, the "narrow" N-terminal region can be expressed as the region from the N-terminus to the start of the intermediate region, i.e., from the N-terminus to the start of the amino acid sequence encoded by exon 2 or exon 3 of the MAPT gene.

[0045] The phosphorylated tau protein of the present disclosure may be a tau protein including the six isoforms described above in which at least one amino acid residue is phosphorylated. Representative examples include tau 441 protein in which at least one of the amino acids at positions 46, 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, 238, 262, and 356 is phosphorylated. These amino acid residues are either serine or threonine.

[0046] Among these, representative phosphorylated tau proteins are those in which at least one of the amino acid residues at positions 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, and 238 is phosphorylated. These amino acid residues are contained in the proline-rich region (aa149-244). Therefore, an anti-phosphorylated tau antibody capable of recognizing phosphorylation of these amino acid residues corresponds to a first antibody that recognizes the intermediate region. For example, in the Examples described below, an anti-phosphorylated tau antibody (first antibody) in which the 181st amino acid residue (threonine) is phosphorylated is used.

[0047] Note that the tau protein and phosphorylated tau protein in the present disclosure are not limited to the six isoforms described above, and may be isoforms other than these (for example, unknown isoforms). In other words, the tau protein or phosphorylated tau protein to be detected in the present disclosure may be a gene product transcribed and translated (and post-translationally modified as necessary) from the MAPT gene, and is not limited to known isoforms.

[0048] [Tau antibody] The tau antibody used in the tau protein detection method according to the present disclosure is an antibody that reacts with the above-described tau protein or phosphorylated tau protein as an antigen. As described above, the present disclosure uses two types of tau antibodies in combination: a first antibody that is used immobilized on a carrier or labeled with a molecule capable of binding to the carrier, and a second antibody that is used labeled without being immobilized on a carrier.

[0049] The carrier on which the first antibody is immobilized is not particularly limited, and any carrier known in the field of antigen-antibody reactions can be suitably used. Specific examples include magnetic beads, resin beads, glass beads, resin plates, membranes, and resin tubes. The type of resin material (polymer compound) used for resin beads, resin plates, resin tubes, and the like is not particularly limited, and any resin material known in the field of antigen-antibody reactions can be suitably used.

[0050] The specific configuration of the magnetic beads is not particularly limited, and examples thereof include a configuration in which the surface of particulate magnetic material is coated with a resin material. The material used for the membrane is also not particularly limited, and publicly known materials such as nitrocellulose or polyvinylidene fluoride (PVDF) can be used. The specific configuration (shape, dimensions, etc.) of the beads, plate, membrane, or tube is also not particularly limited, and any known configuration deemed suitable for a tau protein detection method may be used.

[0051] The method for immobilizing (immobilizing) the first antibody on a carrier is not particularly limited, and any method known in the field of antigen-antibody reactions can be suitably used. Alternatively, the first antibody can be immobilized on a suitable carrier at an appropriate time using a commercially available antibody immobilization kit.

[0052] The first antibody may not be directly immobilized on a carrier, but may be an antibody labeled with a molecule capable of binding to a carrier. For example, the first antibody may be labeled with a labeling molecule such as biotin or avidin, and the first antibody may be immobilized on the carrier by binding to the labeling molecule. Therefore, the first antibody in this embodiment includes not only an antibody directly immobilized on a carrier, but also an antibody indirectly immobilized via a molecule capable of binding to a carrier. Therefore, in this embodiment, the first antibody can be described as an antibody substantially immobilized on a carrier. Note that the labeling molecule capable of binding to a carrier is not limited to biotin or avidin, and various known molecules can be used.

[0053] The label applied to the second antibody is not particularly limited, and any label known in the field of antigen-antibody reactions can be suitably used. Specific examples include enzymes, fluorescent dyes, fluorescent proteins, and biotin. Furthermore, the method for labeling the second antibody is not particularly limited, and any method known in the field of antigen-antibody reactions can be suitably used. Alternatively, the second antibody may be labeled with a suitable type of label at any time using a commercially available antibody labeling kit.

[0054] In the present disclosure, the epitopes recognized by the first and second antibodies, respectively, may be any amino acid sequence contained in the intermediate region or the N-terminal region of tau protein or phosphorylated tau protein. In other words, the first and second antibodies may not be tau antibodies that recognize an amino acid sequence contained in the C-terminal region of tau protein or phosphorylated tau protein as an epitope.

[0055] Therefore, the first antibody may be a tau antibody having an epitope consisting of an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein, or a tau antibody having an epitope consisting of an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein.Similarly, the second antibody may be a tau antibody having an epitope consisting of an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein, or a tau antibody having an epitope consisting of an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein.

[0056] In the examples described below, (1) an antibody having an epitope that is an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein is used as the first antibody, and (2) an antibody having an epitope that is an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein, or (3) an antibody having an epitope that is an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein is used as the second antibody, but it goes without saying that the present disclosure is not limited to such a combination of tau antibodies (1) and (2) or a combination of tau antibodies (1) and (3). For example, the first antibody may be a tau antibody having an epitope that is an amino acid sequence contained in the N-terminal region.

[0057] The method for producing (manufacturing) these first and second antibodies, i.e., the method for producing antibodies having an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein as an epitope, and the method for producing antibodies having an amino acid sequence contained in the N-terminal region of tau protein or phosphorylated tau protein as an epitope, are not particularly limited, and they may be produced by known methods using tau protein or phosphorylated tau protein as the antigen.

[0058] For ease of explanation, a tau antibody having an epitope consisting of an amino acid sequence contained in the intermediate region may be referred to as a "tau antibody recognizing the intermediate region," and a tau antibody having an epitope consisting of an amino acid sequence contained in the N-terminal region may be referred to as a "tau antibody recognizing the N-terminal region." Furthermore, for ease of explanation, a tau antibody having an epitope consisting of an amino acid sequence contained in the C-terminal region, which is not used as a first or second antibody in the present disclosure, may also be referred to as a "tau antibody recognizing the C-terminal region."

[0059] The tau protein or phosphorylated tau protein used as the antigen may be commercially available or may be prepared by known methods using genetic recombination technology. The host cells expressing the tau protein or phosphorylated tau protein may be bacteria (prokaryotic cells) such as Escherichia coli, or eukaryotic cells such as yeast or cultured animal or plant cells. Alternatively, a cell-free protein synthesis system may be used. In the Examples described below, tau protein or phosphorylated tau protein is prepared using yeast as a host according to the method described in Reference 3: Tom Vandebroek et al., "Identification and Isolation of a Hyperphosphorylated, Conformationally Changed Intermediate of Human Protein Tau Expressed in Yeast," BIOCHEMISTRY, Volume 44, 2005, pp. 11466-11475.

[0060] Known methods for producing tau antibodies (tau antibodies recognizing the intermediate region and tau antibodies recognizing the N-terminal region) using such antigens include, for example, the method described in Reference 4: Saeed Zarei t. al., "Production and Characterization of a Peptide-Based Monoclonal Antibody Against CD44 Variant 6," MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, Volume 34, 2015, pp. 36-42. In the Examples described below, seven types of tau antibodies (including the tau antibody recognizing the C-terminal region used in the Comparative Examples) were produced according to the method described in Reference 4.

[0061] In the method described in Reference 4, mice are immunized with a conjugated protein in which the antigen, tau protein or phosphorylated tau protein, is covalently bound to KLH (Keyhole Limpet Hemocyanin) to produce hybridomas from the mouse spleen. These hybridomas are then cultured, and tau antibodies are extracted from the culture supernatant. The recognition sites of the obtained tau antibodies can be identified using peptides having the corresponding amino acid sequences.

[0062] Furthermore, since commercially available tau antibodies are known, in the tau protein detection method according to the present disclosure, if tau antibodies corresponding to the intermediate region-recognizing tau antibody and the N-terminal region-recognizing tau antibody are commercially available, both commercially available antibodies may be used. Alternatively, if one of these tau antibodies is commercially available and the other is not (for example, if the intermediate region-recognizing tau antibody is commercially available and the N-terminal region-recognizing tau antibody is not commercially available, or if the intermediate region-recognizing tau antibody used as the first antibody is commercially available and the intermediate region-recognizing tau antibody used as the second antibody is not commercially available), a commercially available antibody may be used in combination with a tau antibody prepared as described above.

[0063] The intermediate region-recognizing tau antibodies used as the first or second antibodies in the present disclosure have as their epitope (recognition sequence) an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein. Therefore, the epitope of the intermediate region-recognizing tau antibody may be an amino acid sequence contained in the "broad" intermediate region consisting of amino acids 45 to 372 (aa45-372) based on tau441 protein, or an amino acid sequence contained in the standard intermediate region consisting of amino acids 149 to 372 (aa149-372) (a region consisting only of the proline-rich region and microtubule-binding region).

[0064] Furthermore, if the intermediate region-recognizing tau antibody is an anti-phosphorylated tau antibody that recognizes phosphorylated tau protein, the phosphorylation site (position of the phosphorylated amino acid residue) of tau protein recognized by the intermediate region-recognizing tau antibody may be included in the "broad" intermediate region or the standard intermediate region, as described above. Preferably, the phosphorylation site recognized by the intermediate region-recognizing tau antibody is an amino acid sequence included in the proline-rich region (amino acid sequence from positions 149 to 244, aa149-244).

[0065] The N-terminal region-recognizing tau antibody used as the first or second antibody in the present disclosure has an epitope that is an amino acid sequence contained in the N-terminal region of tau protein. Therefore, the epitope of the N-terminal region-recognizing tau antibody may be an amino acid sequence contained in the standard N-terminal region consisting of the amino acid sequence from positions 1 to 148 (aa1-148), or may be an amino acid sequence contained in the "narrowly defined" N-terminal region consisting of the amino acid sequence from positions 1 to 44 (aa1-44).

[0066] In the tau protein detection method according to the present disclosure, the epitopes of the intermediate region-recognizing tau antibodies and the N-terminal region-recognizing tau antibodies have more preferred ranges (preferred regions) in the intermediate region or the N-terminal region. Specifically, the amino acid sequence serving as the epitope of the intermediate region-recognizing tau antibodies is preferably contained in the amino acid sequence from 181 to 191 of the intermediate region (aa181-191) or from 218 to 225 of the amino acid sequence (aa218-225) of the intermediate region. Furthermore, the amino acid sequence serving as the epitope of the N-terminal region-recognizing tau antibodies is preferably contained in the amino acid sequence from 1 to 20 of the N-terminal region (aa1-20) or from 16 to 24 of the N-terminal region (aa16-24). When the intermediate region-recognizing tau antibodies and the N-terminal region-recognizing tau antibodies have an amino acid sequence contained in the preferred region as their epitopes, the detection accuracy of tau protein or phosphorylated tau protein can be further improved.

[0067] [Tau protein detection method] The tau protein detection method according to the present disclosure uses a blood sample as a specimen, reacts the specimen with two types of antibodies, the first and second antibodies described above, and detects tau protein or phosphorylated tau protein in the specimen. Here, in the present disclosure, as described above, the first and second antibodies do not use tau antibodies with epitopes in the C-terminal region of tau protein or phosphorylated tau protein, but use antibodies with epitopes in the intermediate region or N-terminal region. Furthermore, a non-immobilized support (a non-capture support that does not immobilize an antibody as a capture molecule) is not used in addition to a solid-phase support (a capture support that immobilizes an antibody as a capture molecule) that immobilizes the first antibody. This allows for good detection of tau protein or phosphorylated tau protein contained in exosomes in a blood sample.

[0068] In the examples of Patent Document 2, a commercially available antibody that recognizes the amino acid sequence from positions 159 to 163 (aa159-163) of tau protein is used as the "capture antibody," and a commercially available antibody that recognizes phosphorylation of the 181st amino acid (threonine) residue of phosphorylated tau protein is used as the "detection antibody." The capture antibody is bound to capture beads, and this capture antibody is first bound to the target protein (phosphorylated tau protein). The detection antibody is then bound to the target protein bound to the capture antibody, forming an immune complex of capture antibody-target protein-detection antibody.

[0069] The capture antibody is used in a state bound to capture beads and recognizes the amino acid sequence of aa159-163 of tau protein, and therefore corresponds to the first antibody and mid-region-recognizing tau antibody in the present disclosure. On the other hand, the detection antibody is an antibody labeled with biotin that is not bound to capture beads and recognizes phosphorylation at amino acid residue 181 of phosphorylated tau protein, and therefore corresponds to the second antibody and mid-region-recognizing tau antibody in the present disclosure.

[0070] In Patent Document 2, both in the examples and in the embodiments, a capture antibody corresponding to the first antibody in the present disclosure is reacted with phosphorylated tau protein first, and then a detection antibody corresponding to the second antibody is reacted with phosphorylated tau protein. In Patent Document 2, two types of antibodies are reacted with tau protein in this order, and therefore, although the reason is unclear, it is thought that a large amount of non-capture beads (non-immobilized carriers that do not immobilize antibodies) must be used in combination.

[0071] Furthermore, the examples in Patent Document 2 only show the results of distinguishing between AD patients and control patients, and do not specifically disclose the accuracy of tau protein detection, etc. In particular, it has been shown that a small amount of non-capture beads makes it impossible to sufficiently distinguish between AD patients. This suggests that the method described in Patent Document 2 is unable to sufficiently detect phosphorylated tau protein, even to the extent that it is unable to sufficiently detect tau protein contained in exosomes.

[0072] In contrast, with the tau protein detection method according to the present disclosure, as is clear from the comparative results of the Examples and Reference Examples described below, a significant correlation is observed between the detection results of tau protein or phosphorylated tau protein detected from exosomes extracted from blood samples and the detection results of tau protein or phosphorylated tau protein detected from the blood sample itself (Figures 2 to 5). Furthermore, as is clear from the results of the Examples and Comparative Examples, the tau protein detection method according to the present disclosure achieves extremely high detection accuracy, with a sensitivity of 70% or more, a specificity of 70% or more, and an ROC curve (Receiver Operating Characteristic curve) area of 0.70 or more.

[0073] Therefore, in the tau protein detection method according to the present disclosure, tau antibody having an epitope in the C-terminal region is not used, and only a solid-phase support for immobilizing the first antibody is used, without using a non-solid-phase support (non-capture support) that does not immobilize the antibody, thereby enabling detection of tau protein or phosphorylated tau protein with good detection accuracy.

[0074] Furthermore, the tau protein detection method according to the present disclosure makes it possible to detect tau protein or phosphorylated tau protein contained in exosomes using a blood sample as a specimen before exosome extraction, without extracting exosomes. This effectively avoids the possibility of loss of tau protein during the exosome extraction process, allowing for detection of tau protein (or phosphorylated tau protein) with higher sensitivity, and effectively suppresses or avoids the complexity of tau protein detection.

[0075] As described above, the specimen used in the tau protein detection method according to the present disclosure is not particularly limited as long as it is a blood sample. Specific examples of blood samples include whole blood, plasma, and serum. Among these, plasma is more preferred. The method for separating plasma from whole blood is not particularly limited, and plasma separated from whole blood collected by a known blood collection method using an anticoagulant such as heparin salt, citrate salt, or ethylenediaminetetraacetic acid (EDTA) salt may be used.

[0076] As described above, the tau protein detection method according to the present disclosure uses a tau antibody with an optimized recognition sequence (epitope) for tau protein or phosphorylated tau protein, and uses a first antibody substantially immobilized on a carrier in combination with a labeled second antibody as the tau antibody, and reacts the first antibody and the second antibody with a blood sample as a specimen, so long as the method is such that other specific treatments, treatment conditions, and the order of treatments are not particularly limited.

[0077] In the examples described below, a blood sample as a specimen is appropriately diluted and first reacted with a second antibody, and then reacted with a first antibody, after which the carrier is separated and washed, but the specific methods, conditions, reagents used, etc. for such an order of reaction of the first and second antibodies, dilution of the blood sample, separation of the carrier, washing of the immune complex, etc. are not particularly limited. As long as tau protein or phosphorylated tau protein can be detected satisfactorily, the order of reaction of the first and second antibodies may be selected appropriately, some of the processes such as dilution, separation, and washing may be omitted, processes other than these processes may be performed, the order of these processes may be reversed, the same process or different reagents may be used, or the same process may be repeated multiple times.

[0078] The specific method for detecting tau protein or phosphorylated tau protein is not particularly limited. In the examples described below, a luminescent substrate is added to the washed carrier, and the reaction is allowed to proceed, and the amount of luminescence (luminescence intensity) is measured using a commercially available photometer to detect tau protein or phosphorylated tau protein. However, the detection of tau protein or phosphorylated tau protein is not limited to such a method that involves measuring the amount of luminescence, and other known methods may also be used. For example, tau protein or phosphorylated tau protein may be detected using a known detection kit.

[0079] The tau protein detection method according to the present disclosure can effectively detect tau protein or phosphorylated tau protein, and therefore can be suitably used for identifying (differentiating or diagnosing) neurodegenerative diseases. That is, the present disclosure may include a method for identifying neurodegenerative diseases in a human (or mammal) who is the sample collector (subject) by detecting tau protein or phosphorylated tau protein in a blood sample that is the sample using the above-described tau protein detection method.

[0080] Therefore, the specimen used in the tau protein detection method according to the present disclosure may be a blood sample obtained from a subject suspected of having a neurodegenerative disease. This subject may be not only a person who has developed a neurodegenerative disease or a person who shows symptoms that suggest the onset of the disease, but also a person who does not show any symptoms but whose possibility of having a neurodegenerative disease is to be determined, i.e., a healthy person.

[0081] The neurodegenerative diseases to be identified by the tau protein detection method according to the present disclosure may be any diseases that develop (or have the potential to develop, or are suggested to develop) due in part to abnormalities in tau protein. Diseases that are thought to be primarily caused by abnormal accumulation of phosphorylated tau protein are collectively known as "tauopathies." The tau protein detection method according to the present disclosure can be suitably used to identify such tauopathies. Specific examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies. [Example]

[0082] The present invention will be described in more detail based on examples, comparative examples, and reference examples, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention. The antibodies, samples, reagents, etc., and detection methods, etc. used in the following examples, etc., were as follows.

[0083] (Antibodies, specimens, reagents, etc., and detection methods) [Tau antibody] The tau antibodies used in the following Examples, Comparative Examples, and Reference Examples are as shown in Table 1. These tau antibodies were prepared according to the method described in Reference 4. The tau protein or phosphorylated tau protein used as the antigen was prepared according to the method described in Reference 3, as described above. The recognition site of each tau antibody was identified using a peptide having the corresponding amino acid sequence.

[0084] [Table 1]

[0085] [Specimen] In the examples and comparative examples, a total of 20 types of plasma were used as specimens: 10 types of plasma provided by 10 AD patients and 10 types of plasma provided by 10 healthy individuals. In the reference examples, 20 types of exosomes (exosome extracts) were extracted from these plasma samples by polymer precipitation.

[0086] [Reagents, etc.] The diluent used for diluting the sample was phosphate buffer containing 2.0% bovine serum albumin. The wash solution used was phosphate buffer containing 0.05% Tween 20. The luminescent substrate used was Lumigen APS-5 manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0087] The carrier used to immobilize the primary antibody was dynabead magnetic particles (magnetic beads) manufactured by Thermo Electronics, Inc. The primary antibody was immobilized on the magnetic particles according to the method described in the dynabead package insert.

[0088] The secondary antibody was labeled with purified calf intestinal alkaline phosphatase (ALP) manufactured by Thermo Scientific Co. The secondary antibody was labeled with ALP using the Alkaline Phosphatase Labeling Kit manufactured by Dojindo Chemical Industries, Ltd., according to the method described in the attached instructions.

[0089] The amount of luminescence was measured using a photometer named CL-JACK NX manufactured by Hitachi Chemical Diagnostics Systems.

[0090] [Tau protein detection method] A diluted sample was prepared by mixing 1 volume part of human plasma (Example or Comparative Example) or exosome extract (Reference Example) with 1 volume part of diluent. Two volumes of this diluted sample were further mixed with 2 volumes of diluent and 4 volumes of ALP-labeled antibody solution as the second antibody. This prepared a first reaction solution, which was then allowed to react at 37°C for a predetermined time.

[0091] Four parts by volume of magnetic particles with the first antibody immobilized thereon were added to eight parts by volume of the first reaction solution after the reaction and mixed to prepare a second reaction solution, which was then reacted at 37°C for a predetermined time.

[0092] After the reaction, the magnetic particles were separated from the second reaction solution and washed with a washing solution. Eight parts by volume of a luminescent substrate was added to the washed magnetic particles, and the reaction was allowed to proceed at 25°C for several to several tens of seconds. The luminescence intensity (amount of luminescence) was measured to detect tau protein or phosphorylated tau protein.

[0093] Based on the results of detecting tau protein or phosphorylated tau protein from 20 types of samples, the sensitivity and specificity were calculated for each Example, Comparative Example, or Reference Example, and an ROC curve was created, and the area of the ROC curve (ROC area) was calculated. In the Examples and Comparative Examples, a sensitivity of 70% or more, a specificity of 70% or more, and an ROC area of 0.70 or more were considered to be sufficiently accurate. Furthermore, since the Reference Example using exosome extracts is an experimental result for comparison with the Examples or Comparative Examples, only a graph of the comparison results is shown, and the results of sensitivity, etc. are not described.

[0094] Example 1 Antibody T4, a tau antibody recognizing the middle region of tau 441 protein that recognizes the amino acid sequence from positions 218 to 225 (aa218-225), was used as the first antibody, and antibody T1, a tau antibody recognizing the N-terminal region of tau 441 protein that recognizes the amino acid sequence from positions 1 to 20 (aa1-20), was used as the second antibody.Tau protein was detected by measuring the amount of luminescence for 20 types of samples (plasma) as described above.

[0095] Based on the results of detecting tau protein, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody in this Example 1. The results are shown in Table 2.

[0096] Example 2 Tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 1, except that antibody T2, an N-terminal region-recognizing tau antibody that recognizes the amino acid sequence from positions 16 to 24 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in this Example 2 were evaluated. The results are shown in Table 2.

[0097] Furthermore, the detection results of Example 2 and Reference Example 1 were plotted for comparison, with the luminescence intensity as the detection result of this Example 2 on the vertical axis and the luminescence intensity as the detection result of Reference Example 1 described below on the horizontal axis. The results are shown in Figure 2.

[0098] Example 3 Tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 1, except that antibody T3, a tau antibody recognizing the middle region of the tau 441 protein that recognizes the amino acid sequence from positions 185 to 191, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in this Example 3 were evaluated. The results are shown in Table 2.

[0099] Furthermore, the detection results of Example 3 and Reference Example 2 were plotted for comparison, with the luminescence intensity as the detection result of this Example 3 on the vertical axis and the luminescence intensity as the detection result of Reference Example 2 described below on the horizontal axis. The results are shown in Figure 3.

[0100] Example 4 Tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 1, except that antibody T4, i.e., the same antibody as the first antibody, was used as the second antibody, which was an intermediate region-recognizing tau antibody. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody in this Example 4. The results are shown in Table 2.

[0101] (Comparative Example 1) Tau protein was measured by detecting the amount of luminescence in 20 types of specimens (plasma) as described above in the same manner as in Example 1, except that antibody T5, a comparative antibody that recognizes the amino acid sequence from positions 395 to 417 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Comparative Example 1 were evaluated. The results are shown in Table 2.

[0102] The detection results of Comparative Example 1 and Reference Example 3 were plotted for comparison, with the luminescence intensity as the detection result of Comparative Example 1 on the vertical axis and the luminescence intensity as the detection result of Reference Example 3 (described later) on the horizontal axis. The results are shown in Figure 4.

[0103] (Comparative Example 2) Tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 1, except that antibody T6, a comparative antibody that recognizes the amino acid sequence from positions 428 to 441 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Comparative Example 2 were evaluated. The results are shown in Table 2.

[0104] (Reference example 1) Tau protein was detected by measuring the luminescence intensity as described above for 20 types of specimens (plasma) in the same manner as in Example 2, except that the above-mentioned exosome extract was used as the specimen instead of plasma.

[0105] The luminescence intensity as the detection result of Example 2 was plotted on the vertical axis and the luminescence intensity as the detection result of Reference Example 1 on the horizontal axis, and the detection results of Example 2 and Reference Example 1 were plotted for comparison. The results are shown in Figure 2.

[0106] (Reference example 2) Tau protein was detected by measuring the luminescence intensity as described above for 20 types of specimens (plasma) in the same manner as in Example 3, except that the above-mentioned exosome extract was used as the specimen instead of plasma.

[0107] The luminescence intensity as the detection result of Example 3 was plotted on the vertical axis and the luminescence intensity as the detection result of Reference Example 2 on the horizontal axis, and the detection results of Example 3 and Reference Example 2 were plotted for comparison. The results are shown in Figure 3.

[0108] (Reference example 3) Tau protein was detected by measuring the luminescence intensity as described above for 20 types of specimens (plasma) in the same manner as in Comparative Example 1, except that the above-mentioned exosome extract was used instead of plasma as the specimen.

[0109] The detection results of Comparative Example 1 and Reference Example 3 were plotted for comparison, with the luminescence intensity as the detection result of Comparative Example 1 on the vertical axis and the luminescence intensity as the detection result of Reference Example 3 on the horizontal axis. The results are shown in Figure 4.

[0110] [Table 2]

[0111] Example 5 As the first antibody, antibody Tp, a tau antibody recognizing the intermediate region, was used, which recognizes phosphorylated tau protein (pT181) in which the 181st amino acid (threonine) of tau 441 protein is phosphorylated as an antigen (i.e., recognizes the phosphorylation site present in the intermediate region).As the second antibody, antibody T1, a tau antibody recognizing the N-terminal region, which recognizes the amino acid sequence from the 1st to the 20th amino acids of tau 441 protein, was used.The phosphorylated tau protein was detected by measuring the amount of luminescence for 20 types of samples (plasma) as described above.

[0112] Based on the results of detecting phosphorylated tau protein, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody in this Example 5. The results are shown in Table 3.

[0113] Example 6 Phosphorylated tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 5, except that antibody T2, an N-terminal region-recognizing tau antibody that recognizes the amino acid sequence from positions 16 to 24 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Example 6 were evaluated. The results are shown in Table 3.

[0114] Example 7 Phosphorylated tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 5, except that antibody T3, a tau antibody recognizing the middle region of the tau 441 protein that recognizes the amino acid sequence from positions 185 to 191, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in this Example 7 were evaluated. The results are shown in Table 3.

[0115] Example 8 Phosphorylated tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 5, except that antibody T4, i.e., an intermediate region-recognizing tau antibody that recognizes the amino acid sequence from positions 218 to 225 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in this Example 8 were evaluated. The results are shown in Table 3.

[0116] Furthermore, the detection results of Example 8 and Reference Example 4 were plotted for comparison, with the luminescence intensity as the detection result of this Example 8 on the vertical axis and the luminescence intensity as the detection result of Reference Example 4 (described later) on the horizontal axis. The results are shown in Figure 5.

[0117] (Comparative Example 3) Phosphorylated tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 5, except that antibody T5, a comparative antibody that recognizes the amino acid sequence from positions 395 to 417 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Comparative Example 3 were evaluated. The results are shown in Table 2.

[0118] Comparative Example 4 Phosphorylated tau protein was detected by measuring the luminescence intensity for 20 types of specimens (plasma) as described above in the same manner as in Example 5, except that antibody T6, a comparative antibody that recognizes the amino acid sequence from positions 428 to 441 of tau 441 protein, was used as the second antibody. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Comparative Example 4 were evaluated. The results are shown in Table 2.

[0119] (Reference example 4) The same procedure as in Example 8 was repeated except that the above-mentioned exosome extract was used as the sample, and the luminescence intensity was measured as described above for 20 types of samples (plasma) to detect phosphorylated tau protein.

[0120] The detection results of Example 8 and Reference Example 4 were plotted for comparison, with the luminescence intensity as the detection result of Example 8 on the vertical axis and the luminescence intensity as the detection result of Reference Example 4 on the horizontal axis. The results are shown in Figure 5.

[0121] [Table 3]

[0122] (Comparison of Examples, Comparative Examples, and Reference Examples) As is clear from the results of Examples 1 to 4 and Comparative Examples 1 and 2, the tau protein detection method according to the present disclosure can detect tau protein with good detection accuracy. Similarly, as is clear from the results of Examples 5 to 8 and Comparative Examples 3 and 4, the tau protein detection method according to the present disclosure can detect phosphorylated tau protein with good detection accuracy.

[0123] Furthermore, as shown in Figures 2 to 5, a significant correlation was found between Example 2, Example 3, Comparative Example 1, and Example 8 and Reference Examples 1 to 4, which show results using exosome extracts (straight lines in the figures). Therefore, it is believed that the detection method according to the present disclosure, which uses a blood sample such as plasma as a specimen, adequately reflects the detection results from exosome extracts. Therefore, according to the present disclosure, it is possible to effectively avoid the possibility of loss of tau protein during the exosome extraction process and effectively detect tau protein or phosphorylated tau protein.

[0124] Note that reference examples using exosome extracts were conducted to correspond to all of Examples 1 to 8 and Comparative Examples 1 to 4, but in the detailed explanation of the present invention, reference examples are given only for representative Examples or Comparative Examples to illustrate the comparative relationships (Example 2 is an example of an N-terminal region-recognizing tau antibody (T2) having an epitope in the N-terminal region, Example 3 is an example of an intermediate region-recognizing tau antibody (T3) having an epitope in the intermediate region, Comparative Example 1 is an example of a comparative antibody (T5) having an epitope in the C-terminal region, and Example 8 is an example of another intermediate region-recognizing tau antibody (T4) having an epitope in the intermediate region).

[0125] For ease of explanation, comparison results for all Examples and Comparative Examples are not shown, but a significant correlation was found between all Examples and Comparative Examples and the Reference Example. Comparative Examples 1 to 4 have inferior detection accuracy compared to Examples 1 to 8 because the second antibody recognizes the amino acid sequence of the C-terminal region, but are similar to Examples 1 to 8 in that they well reflect the detection results from exosome extracts.

[0126] Thus, the tau protein detection method according to the present disclosure is a tau protein detection method that uses two types of antibodies that specifically bind to tau protein or phosphorylated tau protein to detect tau protein or phosphorylated tau protein from a specimen collected from a subject through an antigen-antibody reaction, wherein the specimen is a blood sample, one of the two types of antibodies is a first antibody that is used by being immobilized on a carrier or is labeled with a molecule that can bind to the carrier, and the other is a second antibody that is not immobilized on a carrier and is labeled, and when the tau protein is divided into an N-terminal region, a C-terminal region, and an intermediate region located between these, the epitopes recognized by the first antibody and the second antibody are amino acid sequences contained in the intermediate region or amino acid sequences contained in the N-terminal region, and one of the two types of antibodies is first subjected to an antigen-antibody reaction with the blood sample, and then the other is subjected to an antigen-antibody reaction with the blood sample.

[0127] According to this configuration, tau protein or phosphorylated tau protein in a sample is detected by a sandwich method that combines a substantially immobilized first antibody with a non-immobilized, labeled second antibody. In this case, antibodies having an epitope in the intermediate region or N-terminal region of tau protein are used as the first and second antibodies, rather than antibodies having an epitope in the C-terminal region of tau protein. Furthermore, a non-immobilized support on which no antibody is immobilized (a non-capture support on which no antibody is immobilized as a capture molecule) is not used in addition to a solid-phase support on which the first antibody is immobilized (a capture support on which an antibody serving as a capture molecule is immobilized).

[0128] This method enables the effective detection of tau protein or phosphorylated tau protein contained in exosomes from blood samples without pretreatment such as exosome extraction. This effectively avoids the possibility of tau protein loss during the exosome extraction process, allowing for more sensitive detection of tau protein (or phosphorylated tau protein). Furthermore, not only does it eliminate the need for exosome extraction, but it also eliminates the need to prepare a non-immobilized support or adjust the ratio of the non-immobilized support to the immobilized support. This effectively reduces or avoids the complexity of tau protein detection. Furthermore, since antibodies that bind to the C-terminal region do not need to be used as the first and second antibodies, the flexibility in selecting the two types of antibodies is improved.

[0129] The present invention is not limited to the description of the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or multiple modified examples are also included in the technical scope of the present invention.

[0130] Furthermore, many modifications and other embodiments of the present invention will be apparent to those skilled in the art from the above description. Therefore, the above description should be construed as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode for carrying out the present invention. Details of the structure and / or function thereof can be substantially changed without departing from the spirit of the present invention. [Industrial Applicability]

[0131] The present invention can be widely and suitably used not only in the field of detecting tau protein or phosphorylated tau protein from a sample, but also in various application fields that utilize the detection of tau protein or phosphorylated tau protein.

Claims

1. A method for detecting phosphorylated tau protein in a sample collected from a subject by antigen-antibody reaction using two types of antibodies that specifically bind to tau protein or phosphorylated tau protein, comprising: the sample is whole blood or plasma; one of the two types of antibodies is a first antibody that is used by being immobilized on a carrier or is labeled with a molecule that can bind to the carrier, and the other is a second antibody that is not immobilized on the carrier and is labeled; When the tau protein or the phosphorylated tau protein is divided into an N-terminal region, a C-terminal region, and an intermediate region located therebetween, the first antibody is an antibody that recognizes an amino acid sequence contained in the intermediate region of the phosphorylated tau protein as an epitope, and the second antibody is an antibody that recognizes an amino acid sequence contained in the intermediate region or an amino acid sequence contained in the N-terminal region as an epitope; the phosphorylated tau protein is detected from the specimen by first subjecting one of the two types of antibodies to an antigen-antibody reaction with the specimen, and then subjecting the other antibody to an antigen-antibody reaction with the specimen, thereby forming an immune complex immobilized on the carrier, without using non-capture beads that do not have an antibody that binds to the phosphorylated tau protein in combination; Method for detecting phosphorylated tau protein.

2. When the tau 441 protein, which has the longest amino acid sequence of 441 residues among the tau protein isoforms, is used as a standard, the N-terminal region is a region consisting of the 1st to 44th amino acid sequence of the Tau441 protein, the intermediate region is a region consisting of the amino acid sequence from the 103rd to the 371st amino acids of the Tau441 protein, The method for detecting phosphorylated tau protein according to claim 1 .

3. When the amino acid sequence serving as the epitope of the antibody is contained in the intermediate region, the epitope is contained in a proline-rich region consisting of an amino acid sequence from 149th to 244th in the intermediate region. The method for detecting phosphorylated tau protein according to claim 2 .

4. When the amino acid sequence serving as the epitope of the antibody is contained in the intermediate region, the epitope is contained in a proline-rich region consisting of an amino acid sequence from 188th to 244th in the intermediate region. The method for detecting phosphorylated tau protein according to claim 2 .

5. When the tau 441 protein, which has the longest amino acid sequence of 441 residues among the tau protein isoforms, is used as a standard, The phosphorylated tau protein is characterized in that at least one of the amino acid residues at positions 46, 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, 238, 262, and 356 of the tau 441 protein is phosphorylated. The method for detecting phosphorylated tau protein according to any one of claims 1 to 4.

6. the phosphorylated tau protein serving as an antigen for the first antibody is characterized in that at least one of the amino acid residues at positions 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, and 238 of the tau 441 protein is phosphorylated; The method for detecting phosphorylated tau protein according to claim 5 .

7. the carrier on which the first antibody is immobilized is magnetic beads, resin beads, glass beads, a resin plate, a membrane, or a resin tube; The molecule capable of binding to the carrier is biotin or avidin. The method for detecting phosphorylated tau protein according to any one of claims 1 to 6.

8. the label of the second antibody is an enzyme, a fluorescent dye, a fluorescent protein, or biotin; The method for detecting phosphorylated tau protein according to any one of claims 1 to 7.

9. The specimen is a blood sample obtained from a subject suspected of having a neurodegenerative disease. The method for detecting phosphorylated tau protein according to any one of claims 1 to 8.

10. The neurodegenerative disease is at least one of Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies. The method for detecting phosphorylated tau protein according to claim 9 .

11. A method for identifying a neurodegenerative disease, comprising detecting phosphorylated tau protein in the blood sample using the method for detecting phosphorylated tau protein according to any one of claims 1 to 10.

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