Tau protein detection method using blood sample as specimen

Abstract

To provide a method for detecting tau protein or phosphorylated tau protein using a blood sample as a specimen with excellent sensitivity by avoiding or suppressing complications in detection processing.SOLUTION: A tau protein detection method for identifying a neurodegenerative disease uses whole blood or blood plasma as a specimen. A first antibody which can be immobilized on a carrier recognizes an intermediate region of tau protein or phosphorylated tau protein. A second antibody which is not fixed to the carrier recognize the intermediate region or N-terminal region. Without using a non-solid phased carrier (non-captured carrier) which does not immobilize antibodies, one of the antibodies is made to reaction with the specimen first, and then the other antibody is made to light source antibody reaction with the specimen. By detecting an immune complex immobilized on the carrier formed thereby, tau protein or phosphorylated tau protein can be detected from the specimen.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for detecting tau protein using a blood sample as a specimen, and more particularly to a method for detecting tau protein that can preferably detect tau protein contained in exosomes in a blood sample.

Background Art

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

[0003] Tau protein is known as a phosphorylated protein in which a plurality of serines or threonines in the amino acid sequence are phosphorylated. When an abnormality occurs in this phosphorylation, it is considered to be involved in neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies. Therefore, conventionally, the use of tau protein and phosphorylated tau protein as biomarkers for neurodegenerative diseases has been studied.

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

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

[0006] By the way, as a biological sample that is particularly easy to use as a specimen, a blood sample can be mentioned as described in Patent Document 1. However, since the content of tau protein in blood has been a trace amount of several to several tens of picograms per 1 mL, a highly sensitive detection system for tau protein has been studied conventionally. However, in recent years, it has been revealed that a part of tau protein in blood exists in exosomes. Therefore, by extracting and using exosomes from a biological sample as in Patent Document 1, it has become possible to detect tau protein at a level of several hundred picograms per 1 mL of the biological sample.

[0007] On the other hand, a method of directly detecting tau protein from a blood sample without extracting exosomes has also been studied. For example, Patent Document 2 discloses a method of forming an immune complex of a phosphorylated tau protein, a capture antibody, and a detection antibody on capture beads in the presence of non-capture beads and detecting a signal derived from the immune complex. In Patent Document 2, the epitope of the capture antibody and the epitope of the detection antibody are different, and non-capture beads that do not form an immune complex coexist in an amount of 1.5 times or more with respect to 1 capture bead.

[0008] According to Patent Document 2, by forming an immune complex with non-capture beads coexisting in an amount 1.5 times or more that of capture beads, phosphorylated tau protein can be measured (detected) with a sensitivity capable of discriminating between Alzheimer's disease (AD) patients and subject patients. However, there is no particular description about the specific reason why phosphorylated tau protein can be detected (measured) well due to the presence of non-capture beads.

Prior Art Documents

Patent Document

[0009]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] When the present inventors extracted and disrupted exosomes from a blood sample and confirmed the amount of exosomal proteins by electrophoresis, it was possible to confirm the amount of tau protein at the nanogram level per milliliter. Therefore, it was considered that if tau protein contained in exosomes of a blood sample could be detected with high reactivity, the detection accuracy of tau protein could be further improved. However, in the conventional method, the process of extracting vesicles such as exosomes is essential, which complicates the tau protein detection method.

[0011]

[0012] In addition, in the process of extracting exosomes from a blood sample, it is also assumed that a part of the tau protein in the blood sample may be lost. For example, in Patent Document 1, in order to isolate or concentrate vesicles such as exosomes from a biological sample, drugs, antibodies, flow cytometry, etc. are used, but depending on the conditions of isolation or concentration, etc., a part of the tau protein may be lost (partial loss may occur), and this loss of tau protein may affect the detection results. On the other hand, in a method that does not extract exosomes as in Patent Document 2 and uses a blood sample such as plasma as it is, the occurrence of tau protein loss can be avoided. However, in Patent Document 2, in addition to the capture beads for forming immune complexes, it is necessary to use a larger amount of non-capture beads than the capture beads. Therefore, as a tau protein detection method, there is a possibility that the detection process may be complicated because a large amount of non-capture beads are used.

[0013] In addition, in the examples of Patent Document 2, the ratio of non-capturing beads was examined and the discrimination results between AD patients and control patients were shown, but the detection accuracy of tau protein and the like was not specifically shown. Therefore, it is not clear to what extent the method disclosed in Patent Document 2 can detect tau protein with good sensitivity.

[0014] The present invention has been made to solve such problems, and an object of the present invention is to provide a detection method capable of detecting tau protein or phosphorylated tau protein with better sensitivity by using a blood sample as a specimen and avoiding or suppressing complication of detection processing.

Means for Solving the Problems

[0015] The tau protein detection method according to the present invention is a tau protein detection method for detecting tau protein or phosphorylated tau protein from a specimen collected from a subject by an antigen-antibody reaction using two types of antibodies that specifically bind to tau protein or phosphorylated tau protein, wherein the specimen is a blood sample, one of the two types of antibodies is a first antibody that is immobilized on a carrier and used, or a first antibody labeled with a molecule capable of binding 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 between them, the epitopes recognized by the first antibody and the second antibody are amino acid sequences included in the intermediate region or amino acid sequences included 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 combining a substantially immobilized first antibody and a labeled second antibody that is not immobilized. At this time, as the first antibody and the second antibody, an antibody having an epitope in the middle region or the N-terminal region is used instead of an antibody having an epitope in the C-terminal region of the tau protein, and a solid-phase carrier for immobilizing the first antibody (a capture carrier having an antibody as a capture molecule immobilized thereon) is used. Separately, a non-solid-phase carrier that does not immobilize an antibody (a non-capture carrier that does not immobilize an antibody as a capture molecule) is not used in combination.

[0017] Thereby, even without performing pretreatment such as exosome extraction, tau protein or phosphorylated tau protein contained in exosomes of a blood sample can be detected well. Therefore, it is possible to effectively avoid the possibility of loss of tau protein during the exosome extraction process and detect tau protein (or phosphorylated tau protein) with better sensitivity. In addition, not only is the process of extracting exosomes no longer necessary, but also since a non-solid-phase carrier is not used in combination, there is no need to prepare a non-solid-phase carrier or adjust the abundance ratio of the non-solid-phase carrier to the solid-phase carrier. Therefore, it is possible to effectively suppress or avoid the complication of detecting tau protein. Furthermore, as the first antibody and the second antibody, it is only necessary not to use an antibody that binds to the C-terminal region, so the degree of freedom in selecting two types of antibodies can be improved.

[0018] In the method for detecting tau protein having the above configuration, when the N-terminal region is based on the tau441 protein having 441 residues, which is the longest amino acid sequence among the isoforms of the tau protein, the N-terminal region is a region consisting of the amino acid sequence from the 1st to the 44th amino acids of the tau441 protein, and the middle region is a region consisting of the amino acid sequence from the 103rd to the 371st amino acids of the tau441 protein. It may be a configuration.

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

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

[0021] In the tau protein detection method with the above configuration, when taking the tau441 protein having 441 residues with the longest amino acid sequence among the isoforms of the tau protein as a reference, the phosphorylated tau protein may be a configuration in which at least any one of the amino acid residues at the 46th, 175th, 181st, 185th, 198th, 199th, 202nd, 205th, 208th, 210th, 212th, 214th, 217th, 231st, 235th, 237th, 238th, 262nd, and 356th positions of the tau441 protein is phosphorylated.

[0022] In the tau protein detection method with the above configuration, the phosphorylated tau protein that serves as the antigen of the first antibody may be a configuration in which at least any one of the amino acid residues at the 175th, 181st, 185th, 198th, 199th, 202nd, 205th, 208th, 210th, 212th, 214th, 217th, 231st, 235th, 237th, or 238th positions of the tau441 protein is phosphorylated.

[0023] In the tau protein detection method with 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] In addition, in the method for detecting tau protein having the above-described configuration, the label of the secondary antibody may be an enzyme, a fluorescent dye, a fluorescent protein, or biotin.

[0025] In addition, in the method for detecting tau protein having the above-described configuration, the sample may be a blood sample obtained from a subject suspected of having a neurodegenerative disease.

[0026] In addition, in the method for detecting tau protein having the above-described configuration, the neurodegenerative disease may be at least any one of Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies.

[0027] In addition, the present invention may include a method for discriminating a neurodegenerative disease by detecting tau protein or phosphorylated tau protein in the blood sample using the method for detecting tau protein having the above-described configuration.

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

Effect of the Invention

[0029] In the present invention, with the above configuration, it is possible to provide a detection method capable of detecting tau protein or phosphorylated tau protein with better sensitivity by avoiding or suppressing complication of detection processing using a blood sample as a sample.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0031] Hereinafter, representative embodiments of the present invention will be specifically described. The tau protein detection method according to the present disclosure is a method for detecting a tau protein or a phosphorylated tau protein from a specimen collected from a subject by an antigen-antibody reaction using two types of antibodies (an anti-tau antibody or an anti-phosphorylated tau antibody) that specifically bind to the tau protein or the phosphorylated tau protein. A blood sample is used as the specimen, and as the anti-tau antibody or the anti-phosphorylated tau antibody, a first antibody that is immobilized on a carrier and used, or a molecule that can bind to the carrier and is labeled (substantially immobilized), and a second antibody that is labeled and used without being immobilized on the carrier (not immobilized) are used in combination. In the present disclosure, for convenience of explanation, the anti-tau antibody that specifically binds to the tau protein and the anti-phosphorylated tau antibody that specifically binds to the phosphorylated tau protein are collectively simply abbreviated as "tau antibody".

[0032] Here, when the 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 are the amino acid sequences included in the intermediate region or the amino acid sequences included in the N-terminal region. That is, as the first antibody, a tau antibody having an amino acid sequence included in the intermediate region of the tau protein or phosphorylated tau protein as an epitope may be used, or a tau antibody having an amino acid sequence included in the N-terminal region of the tau protein or phosphorylated tau protein as an epitope may be used. Similarly, as the second antibody, a tau antibody having an amino acid sequence included in the N-terminal region of the tau protein or phosphorylated tau protein as an epitope may be used, or a tau antibody having an amino acid sequence included in the intermediate region of the tau protein or phosphorylated tau protein as an epitope may be used.

[0033] In the method for detecting tau protein according to the present disclosure, either one of the two types of tau antibodies may be first subjected to an antigen-antibody reaction with a blood sample, 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 is not previously immobilized with a blood sample and causing an antigen-antibody reaction, and then the first reaction solution and the immobilized first antibody may be subjected to an antigen-antibody reaction. Alternatively, a first reaction solution may be prepared by subjecting a first antibody previously immobilized with a blood sample to an antigen-antibody reaction, and then the first reaction solution and a second antibody that is not immobilized may be subjected to an antigen-antibody reaction.

[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, described in Reference 1: Hasan A.M.M. Almansoub, et. al., "Tau Abnormalities and the Potential Therapy in Alzheimer's Disease", Journal of Alzheimer's Disease 67 (2019) pp.13-33, or described in Reference 2: International Publication No. 2013 / 180238, etc. The phosphorylated tau protein is one in which some of the amino acids constituting the tau protein are phosphorylated.

[0035] As described in Reference 1 or Reference 2, typically six isoforms of the tau protein are known. The MAPT gene encoding the tau protein has 16 exons, and exons 1 to 13 are expressed in the central nervous system. Among these, alternative splicing occurs for exons 2, 3, and 10, resulting in the six isoforms as described above.

[0036] Specifically, on the N-terminal side, due to alternative splicing of exon 2 or exon 3 of the MAPT gene, the N-terminal repeat sequence N may be 0 to 2 (0N to 2N), and due to alternative splicing of exon 10 of the MAPT gene, the repeat sequence R in the microtubule-binding region may be 3 (1R to 3R) or 4 (1R to 4R). As described in Reference 1, due to the differences in these repeat sequences, the six isoforms of the tau protein can be denoted 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) (there is also a notation in which N and R are reversed as in Reference 2). The tau protein to be detected in the present disclosure includes all of these six isoforms.

[0037] In the present disclosure, the amino acid sequence ordinal numbers are defined based on the 2N4R isoform in which alternative splicing does not occur in any of exons 2, 3, and 10, that is, the tau protein 2N4R having the longest amino acid sequence among six isoforms and having 441 residues. In the present embodiment, for convenience of explanation, the tau protein 2N4R is described as the "tau 441 protein" based on the number of amino acid residues.

[0038] The tau 441 protein (2N4R) is described in Reference 1 and, as shown in FIG. 1, can be divided into four regions: an N-terminal region, a proline-rich domain (PRD), a microtubule-binding domain (MBD), and a C-terminal region. The N-terminal region is composed of the amino acid sequence from the 1st to the 148th (aa1-148), the proline-rich region is composed of the amino acid sequence from the 149th to the 244th (aa149-244), the microtubule-binding region is composed of the amino acid sequence from the 245th to the 372nd (aa244-372), and the C-terminal region is composed of the amino acid sequence from the 373rd to the 441st (aa373-441). In FIG. 1, the two repeat sequences N on the N-terminal side are described as "N1" and "N2", respectively, and the four repeat sequences R in the microtubule-binding region are described as "R1", "R2", "R3", and "R4", respectively.

[0039] In the present disclosure, the amino acid sequence of the tau protein recognized by the tau antibody, that is, the amino acid sequence that becomes the epitope of the tau antibody, is included at least in the middle region, and further in the N-terminal region in addition to the middle region. Here, the middle region of the tau protein according to the present disclosure may be any region located between the N-terminal region and the C-terminal region. Therefore, a region including the proline-rich region and the microtubule-binding region, that is, a region composed of the amino acid sequence from the 149th to the 372nd (aa149-372) can be defined as the middle region.

[0040] In the present disclosure, the N-terminal region is defined as a broad region up to before the proline-rich region, but a narrow region from the N-terminus to before the repeat sequence N can be defined as the "narrow sense" N-terminal region. This is because among the isoforms of the tau protein, 0N3R and 0N4R that do not contain the repeat sequence N are included. That is, if the amino acid sequence recognized by the tau antibody is included in the "narrow sense" N-terminal region, it becomes possible to detect the 0N3R and 0N4R isoforms well. Specifically, the "narrow sense" N-terminal region is composed of the amino acid sequence from the 1st to the 44th (aa1-44).

[0041] If the N-terminal region is a "narrow sense" region, the intermediate region located between the N-terminal region and the C-terminal region can be defined as a region that extends to the N-terminal side. In other words, the reference N-terminal region is composed of the amino acid sequence from the 1st to the 148th (aa1-148), but as described above, the "narrow sense" N-terminal region is composed of the amino acid sequence from the 1st to the 44th (aa1-44). Therefore, the "broad sense" intermediate region can be extended to the 45th amino acid residue on the N-terminal side.

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

[0043] Also, the "broad sense" intermediate region and the "narrow sense" N-terminal region in the present disclosure can be expressed by defining them based on the repeat sequence N and the repeat sequence R, rather than based on the ordinal number of the amino acid sequence of the tau441 protein.

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

[0045] The phosphorylated tau protein in the present disclosure may be any one in which at least one amino acid residue of the tau protein including the six isoforms described above is phosphorylated. Typically, examples include those 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 of the tau441 protein are phosphorylated. These amino acid residues are either serine or threonine.

[0046] Among these, typical phosphorylated tau proteins are those in which at least any one of the amino acid residues at positions 175, 181, 185, 198, 199, 202, 205, 208, 210, 212, 214, 217, 231, 235, 237, or 238 is phosphorylated. These amino acid residues are included in the proline-rich region (aa149 - 244). Therefore, anti-phosphorylated tau antibodies that can recognize the phosphorylation of these amino acid residues correspond to primary antibodies that recognize the intermediate region. For example, in the examples described later, as an example of the anti-phosphorylated tau antibody (primary antibody), one 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 only the six isoforms described above, and may be isoforms different from 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 (post-translational modification, etc. as necessary) from the MAPT gene, and is not limited to only known isoforms.

[0048] [Tau antibody] The tau antibody used in the tau protein detection method according to the present disclosure is an antibody that uses the above-described tau protein or phosphorylated tau protein as an antigen. And in the present disclosure, as the tau antibody, as described above, two types are used in combination: a primary antibody fixed to a carrier and used, or a primary antibody labeled with a molecule capable of binding to the carrier, and a secondary antibody labeled and used without being fixed to the carrier.

[0049] The carrier to which the first antibody is immobilized is not particularly limited, and those known in the field of antigen-antibody reactions can be preferably used. Specifically, for example, magnetic beads, resin beads, glass beads, resin plates, membranes, or resin tubes, etc. can be mentioned. The type of resin material (polymer compound) used for resin beads, resin plates, resin tubes, etc. is not particularly limited, and resin materials known in antigen-antibody reactions can be preferably used.

[0050] Also, the specific configuration of the magnetic beads is not particularly limited, and for example, a configuration in which the surface of particulate magnetic substances is coated with a resin material, etc. can be mentioned. The material used for the membrane is not particularly limited, and known materials such as nitrocellulose or polyvinylidene fluoride (PVDF) can be publicly used. The specific configuration (shape, dimensions, etc.) of the beads, plates, membranes, or tubes is not particularly limited, and any known configuration that is judged to be suitable in the tau protein detection method may be used.

[0051] The method for immobilizing (solid-phasing) the first antibody to the carrier is not particularly limited, and methods known in the field of antigen-antibody reactions can be preferably used. Alternatively, a commercially available antibody immobilization kit can be used to immobilize the first antibody to a suitable carrier in a timely manner.

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

[0053] The label applied to the secondary antibody is not particularly limited, and those known in the field of antigen-antibody reactions can be preferably used. Specifically, for example, enzymes, fluorescent dyes, fluorescent proteins, biotin, etc. can be mentioned. Also, the method for labeling the secondary antibody is not particularly limited, and methods known in the field of antigen-antibody reactions can be preferably used. Alternatively, a commercially available antibody labeling kit may be used to label the secondary antibody with a suitable type of label in a timely manner.

[0054] In the present disclosure, the epitopes recognized by the primary antibody and the secondary antibody may be amino acid sequences contained in the intermediate region of the tau protein or phosphorylated tau protein, or amino acid sequences contained in the N-terminal region. That is, the primary antibody and the secondary antibody may be tau antibodies having an amino acid sequence contained in the C-terminal region of the tau protein or phosphorylated tau protein as an epitope.

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

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

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

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

[0059] As the tau protein or phosphorylated tau protein serving as the antigen, commercially available products may be used, or they may be prepared by known methods using genetic recombination techniques. The host cells that express the tau protein or phosphorylated tau protein may be bacteria (prokaryotic cells) such as Escherichia coli, or eukaryotic cells such as yeast or cultured cells of animals and plants. Alternatively, a cell-free protein synthesis system may be used. In the examples described later, 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, tau protein or phosphorylated tau protein is prepared using yeast as the host.

[0060] As a known method for producing tau antibodies (intermediate region-recognizing tau antibodies and N-terminal region-recognizing tau antibodies) using such an antigen, 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 can be cited. In the examples described later, according to the method described in this Reference 4, seven types of tau antibodies (including the C-terminal region-recognizing tau antibody used in the comparative example) are produced.

[0061] In the method described in Reference 4, a mouse is immunized with a conjugate protein in which the antigenic tau protein or phosphorylated tau protein is covalently bound to KLH (Keyhole Limpet Hemocyanin) to produce hybridomas of the mouse spleen, and these hybridomas are cultured to extract tau antibodies from the supernatant of the culture solution. The recognition site of the obtained tau antibody may be identified using a peptide having each corresponding amino acid sequence.

[0062] In addition, since commercially available tau antibodies are known, in the tau protein detection method according to the present disclosure, if tau antibodies corresponding to the middle region recognition tau antibody and the N-terminal region recognition tau antibody are commercially available, either commercially available antibody may be used. Alternatively, when any of these tau antibodies is commercially available and any of them is not commercially available (for example, when the middle region recognition tau antibody is commercially available and the N-terminal region recognition tau antibody is not commercially available, or when the middle region recognition tau antibody used as the primary antibody is commercially available and the middle region recognition tau antibody used as the secondary antibody is not commercially available, etc.), a commercially available antibody and the tau antibody prepared as described above may be used in combination.

[0063] The middle region recognition tau antibody used as the primary antibody or the secondary antibody in the present disclosure has an amino acid sequence contained in the middle region of the tau protein or phosphorylated tau protein as an epitope (recognition sequence). Therefore, the epitope of the middle region recognition tau antibody may be an amino acid sequence contained in the "broad sense" middle region composed of the 45th to 372nd amino acid sequences (aa45-372) based on the tau441 protein, or an amino acid sequence contained in the reference middle region (a region composed only of the proline-rich region and the microtubule-binding region) composed of the 149th to 372nd amino acid sequences (aa149-372).

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

[0065] The N-terminal region-recognizing tau antibody used as the first antibody or the second antibody in the present disclosure has an amino acid sequence included in the N-terminal region of the tau protein as an epitope. Therefore, the epitope of the N-terminal region-recognizing tau antibody may be an amino acid sequence included in the reference N-terminal region composed of the amino acid sequence from the 1st to the 148th (aa1-148), or may be an amino acid sequence included in the "narrow sense" N-terminal region composed of the amino acid sequence from the 1st to the 44th (aa1-44).

[0066] In the tau protein detection method according to the present disclosure, there are more preferable ranges (preferred regions) in the intermediate region or the N-terminal region as the epitopes of the intermediate region-recognizing tau antibody and the N-terminal region-recognizing tau antibody. Specifically, the amino acid sequence serving as the epitope of the intermediate region-recognizing tau antibody is preferably included in the amino acid sequence from the 181st to the 191st (aa181-191) in the intermediate region, or is preferably included in the amino acid sequence from the 218th to the 225th (aa218-225). Further, the amino acid sequence serving as the epitope of the N-terminal region-recognizing tau antibody is preferably included in the amino acid sequence from the 1st to the 20th (aa1-20) in the N-terminal region, or is preferably included in the amino acid sequence from the 16th to the 24th (aa16-24). If the intermediate region-recognizing tau antibody and the N-terminal region-recognizing tau antibody have an amino acid sequence included in the preferred region as an epitope, the detection accuracy of the tau protein or the 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, and reacts two types of the aforementioned first antibody and second antibody with this specimen to detect tau protein or phosphorylated tau protein in the specimen. Here, in the present disclosure, as the first antibody and the second antibody, as described above, a tau antibody having an epitope in the C-terminal region of tau protein or phosphorylated tau protein is not used, and an antibody having an epitope in the middle region or the N-terminal region is used. Also, separately from the solid-phase carrier (capture carrier having an antibody as a capture molecule) on which the first antibody is immobilized, a non-solid-phase carrier (non-capture carrier having no antibody as a capture molecule) on which no antibody is immobilized is not used in combination. Thereby, tau protein or phosphorylated tau protein contained in exosomes of a blood sample can be detected well.

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

[0069] The capture antibody is used in a state of being bound to capture beads and recognizes the amino acid sequence of aa159-163 of tau protein, and thus corresponds to the first antibody and the middle-region recognizing tau antibody in the present disclosure. On the other hand, the detection antibody is an antibody labeled with biotin without being bound to capture beads, and recognizes phosphorylation of the 181st amino acid residue of phosphorylated tau protein, and thus corresponds to the second antibody and the middle-region recognizing tau antibody in the present disclosure.

[0070] In Patent Document 2, in both the examples and the embodiments, the capture antibody corresponding to the first antibody in the present disclosure is first reacted with the phosphorylated tau protein, and then the detection antibody corresponding to the second antibody is reacted with the phosphorylated tau protein. In Patent Document 2, in order to react two types of antibodies with the tau protein in such an order, although the reason is unclear, it is considered necessary to use a large amount of non-capture beads (non-solid-phase carriers that do not immobilize antibodies) in combination.

[0071] In addition, in the examples of Patent Document 2, only the discrimination results between AD patients and control patients are shown, and the detection accuracy of tau protein and the like is not specifically shown. In particular, it has been shown that when the amount of non-capture beads is small, AD patients cannot be sufficiently discriminated. Therefore, it is suggested that in the method described in Patent Document 2, the detection accuracy of phosphorylated tau protein is such that the tau protein contained in exosomes cannot be sufficiently detected.

[0072] On the other hand, in the tau protein detection method according to the present disclosure, as is clear from the comparison results of the examples and reference examples described later, there is a significant correlation between the detection results of tau protein or phosphorylated tau protein detected from exosomes extracted from a blood sample and the detection results of tau protein or phosphorylated tau protein detected from the blood sample itself (Figs. 2 to 5). Also, as is clear from the results of the examples and comparative examples, in the tau protein detection method according to the present disclosure, a very excellent detection accuracy with a sensitivity of 70% or more, a specificity of 70% or more, and an area of the ROC curve (Receiver Operatorating Characteristic curve) of 0.70 or more is obtained.

[0073] Therefore, in the tau protein detection method according to the present disclosure, in addition to not using a tau antibody having an epitope in the C-terminal region, by using only the solid-phase carrier for immobilizing the first antibody and not using a non-solid-phase carrier (non-capture carrier) that does not immobilize the antibody in combination, tau protein or phosphorylated tau protein can be detected with good detection accuracy.

[0074] In addition, in the tau protein detection method according to the present disclosure, even without extracting exosomes, it is possible to detect tau protein or phosphorylated tau protein contained in exosomes using a blood sample, which is a sample before exosome extraction. Therefore, it is possible to effectively avoid the possibility of loss of tau protein during the exosome extraction process, detect tau protein (or phosphorylated tau protein) with better sensitivity, and effectively suppress or avoid the complication of tau protein detection.

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

[0076] In the tau protein detection method according to the present disclosure, as described above, a tau antibody with an optimized recognition sequence (epitope) of tau protein or phosphorylated tau protein is used. As these tau antibodies, a first antibody substantially immobilized on a carrier and a labeled second antibody are used in combination, and as long as the first antibody and the second antibody are each reacted with a blood sample, which is a sample, other specific treatments, the treatment conditions, and the order of the treatments are not particularly limited.

[0077] In the embodiments described below, a blood sample as a specimen is appropriately diluted and first reacted with a second antibody, and then with a first antibody, and then the carrier is separated and washed. However, the reaction order of the first antibody and the second antibody, specific methods such as dilution of the blood sample, separation of the carrier, washing of the immune complex, conditions, reagents used, etc. are not particularly limited. As long as tau protein or phosphorylated tau protein can be detected well, the reaction order of the first antibody and the second antibody can be appropriately selected, some treatments such as dilution, separation, and washing may be omitted, other treatments other than these may be performed, the order of these treatments may be changed, the same treatment may use different reagents, etc., and the same treatment may be repeated multiple times.

[0078] The specific detection method of tau protein or phosphorylated tau protein is not particularly limited either. In the embodiments described below, a luminescent substrate is added to the washed carrier and reacted, and the luminescence amount (luminescence intensity) is measured with 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 the method by measuring such luminescence amount, and other known methods may be used. For example, a known detection kit may be used to detect tau protein or phosphorylated tau protein.

[0079] The tau protein detection method according to the present disclosure can detect tau protein or phosphorylated tau protein well, so it can be suitably used for the purpose of identifying (distinguishing or diagnosing) neurodegenerative diseases. That is, the present disclosure may include a method for identifying a neurodegenerative disease of a human (or mammal) who is the specimen collector (subject) by detecting tau protein or phosphorylated tau protein in a blood sample as a specimen using the above-described tau protein detection method.

[0080] Therefore, the sample 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 suspected of the onset of the disease, but also a person who has no symptoms but wants to determine whether there is a possibility of having a neurodegenerative disease, that is, a healthy person.

[0081] In the tau protein detection method according to the present disclosure, the neurodegenerative disease to be identified may be a disease that develops (or may develop, or is suggested to develop) with an abnormality of tau protein as one of the factors. Diseases considered to be mainly caused by abnormal accumulation of phosphorylated tau protein are collectively referred to as "tauopathy". The tau protein detection method according to the present disclosure can be suitably used to identify such tauopathy. Specific neurodegenerative diseases include, for example, Alzheimer's disease (AD), frontotemporal dementia (FTD), Lewy body dementia, etc., but are not limited to these diseases.

Examples

[0082] The present invention will be described more specifically 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. In addition, the antibodies, samples, reagents, etc., and detection methods used in the following examples, etc. were carried out as shown below.

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

[0084]

Table 1

[0085] [Specimen] As specimens for the examples and comparative examples, a total of 20 types of plasma were used, including 10 types of plasma provided by 10 AD patients and 10 types of plasma provided by 10 healthy subjects. Also, in the reference example, 20 types of exosomes extracted from these plasmas by the polymer precipitation method (exosome extracts) were used.

[0086] [Reagents, etc.] As a diluent for diluting the specimen, a phosphate buffer containing 2.0% bovine serum albumin was used. As a washing solution, a phosphate buffer containing 0.05% Tween 20 was used. As a luminescent substrate, Lumigen APS-5, a product name of Fujifilm Wako Pure Chemical Corporation, was used.

[0087] As a carrier for immobilizing the first antibody, dynabead magnetic particles (magnetic beads), a product name of Thermo, were used. As a method for immobilizing the first antibody on the magnetic particles, the method described in the dynabead attached document was used.

[0088] As a label for the second antibody, purified calf intestinal alkaline phosphatase (ALP) of Thermo was used. As a method for labeling the second antibody with ALP, the Alkaline Phosphatase Labeling Kit of Dojindo Laboratories was used, and labeling was performed according to the method described in the attached document.

[0089] For measuring the luminescence amount, a CL-JACK NX photometer, a product name of Hitachi Chemical Diagnostics Systems Co., Ltd., was used.

[0090] [Tau protein detection method] One volume part of human plasma (Example or Comparative Example) or exosome extract (Reference Example) as a sample and one volume part of a diluent were mixed to prepare a diluted sample. To 2 volume parts of this diluted sample, 2 volume parts of the diluent and 4 volume parts of an ALP-labeled antibody solution as a second antibody were further added and mixed. Thereby, a first reaction solution was prepared and reacted at 37 °C for a predetermined time.

[0091] To 8 volume parts of the first reaction solution after the reaction, 4 volume parts of magnetic particles immobilized with a first antibody were added and mixed. Thereby, a second reaction solution was prepared and reacted at 37 °C for a predetermined time.

[0092] Magnetic particles were separated from the second reaction solution after the reaction and washed with a washing solution. 8 volume parts of a luminescent substrate were added to the washed magnetic particles and reacted at 25 °C for several seconds to more than ten seconds, and 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, for each Example, Comparative Example, or Reference Example, sensitivity and specificity were calculated and an ROC curve was created, and the area of the ROC curve (ROC area) was calculated. In the Examples and Comparative Examples, when the sensitivity was 70% or more, the specificity was 70% or more, and the ROC area was 0.70 or more, it was evaluated that sufficiently good detection accuracy was obtained. In addition, since the Reference Example using the exosome extract is an experimental result for comparison with the Examples or Comparative Examples, only a graph of the comparison results was illustrated, and the description of the results such as sensitivity was omitted.

[0094] (Example 1) As the first antibody, an intermediate region-recognizing tau antibody that recognizes the amino acid sequence (aa218-225) from the 218th to 225th positions of antibody T4, that is, tau441 protein, was used, and as the second antibody, an N-terminal region-recognizing tau antibody that recognizes the amino acid sequence (aa1-20) from the 1st to 20th positions of antibody T1, that is, tau441 protein, was used. The luminescence amount was measured for 20 types of samples (plasma) as described above to detect tau protein.

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

[0096] (Example 2) Using the same procedure as in Example 1, except that antibody T2, an N-terminal region recognition tau antibody that recognizes the 16th to 24th amino acid sequence of tau441 protein, was used as the second antibody, the luminescence intensity of 20 specimens (plasma) was measured as described above to detect tau protein. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Example 2 were evaluated. The results are shown in Table 2.

[0097] Also, with the luminescence intensity of the detection results of Example 2 as the vertical axis and the luminescence intensity of the detection results of Reference Example 1 described later as the horizontal axis, the detection results of Example 2 and Reference Example 1 were plotted and compared. The results are shown in Figure 2.

[0098] (Example 3) Using the same procedure as in Example 1, except that antibody T3, an intermediate region recognition tau antibody that recognizes the 185th to 191st amino acid sequence of tau441 protein, was used as the second antibody, the luminescence intensity of 20 specimens (plasma) was measured as described above to detect tau protein. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody in Example 3 were evaluated. The results are shown in Table 2.

[0099] Also, with the luminescence intensity of the detection results of Example 3 as the vertical axis and the luminescence intensity of the detection results of Reference Example 2 described later as the horizontal axis, the detection results of Example 3 and Reference Example 2 were plotted and compared. The results are shown in Figure 3.

[0100] (Example 4) As the secondary antibody, except that antibody T4, i.e., the same antibody as the primary antibody, was used as the intermediate region recognition tau antibody, the luminescence amount was measured for 20 specimens (plasma) in the same manner as in Example 1 to detect tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the primary antibody and the secondary antibody of Example 4. The results are shown in Table 2.

[0101] (Comparative Example 1) As the secondary antibody, except that antibody T5, i.e., a comparative antibody that recognizes the amino acid sequence from position 395 to position 417 of tau 441 protein, was used, the luminescence amount was detected for 20 specimens (plasma) in the same manner as in Example 1 to measure tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the primary antibody and the secondary antibody of this Comparative Example 1. The results are shown in Table 2.

[0102] Also, with the luminescence intensity of the detection result of this Comparative Example 1 on the vertical axis and the luminescence intensity of the detection result of Reference Example 3 described later on the horizontal axis, the detection results of these Comparative Example 1 and Reference Example 3 were plotted and compared. The results are shown in Figure 4.

[0103] (Comparative Example 2) As the secondary antibody, except that antibody T6, i.e., a comparative antibody that recognizes the amino acid sequence from position 428 to position 441 of tau 441 protein, was used, the luminescence amount was measured for 20 specimens (plasma) in the same manner as in Example 1 to detect tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the primary antibody and the secondary antibody of this Comparative Example 2. The results are shown in Table 2.

[0104] (Reference Example 1) As the specimen, except that exosome extract described above was used instead of plasma, the luminescence amount was measured for 20 specimens (plasma) in the same manner as in Example 2 to detect tau protein.

[0105] With the luminescence intensity which is the detection result of Example 2 as the vertical axis and the luminescence intensity which is the detection result of Reference Example 1 as the horizontal axis, the detection results of Example 2 and Reference Example 1 were plotted and compared. The result is shown in FIG. 2.

[0106] (Reference Example 2) As a specimen, except that the aforementioned exosome extract instead of plasma was used, the luminescence amount was measured for 20 types of specimens (plasma) in the same manner as in Example 3, and tau protein was detected.

[0107] With the luminescence intensity which is the detection result of Example 3 as the vertical axis and the luminescence intensity which is the detection result of Reference Example 2 as the horizontal axis, the detection results of Example 3 and Reference Example 2 were plotted and compared. The result is shown in FIG. 3.

[0108] (Reference Example 3) As a specimen, except that the aforementioned exosome extract instead of plasma was used, the luminescence amount was measured for 20 types of specimens (plasma) in the same manner as in Comparative Example 1, and tau protein was detected.

[0109] With the luminescence intensity which is the detection result of Comparative Example 1 as the vertical axis and the luminescence intensity which is the detection result of Reference Example 3 as the horizontal axis, the detection results of Comparative Example 1 and Reference Example 3 were plotted and compared. The result is shown in FIG. 4.

[0110]

Table 2

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

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

[0113] (Example 6) As in Example 5, except that an N-terminal region recognition tau antibody that recognizes the amino acid sequence from the 16th to the 24th of antibody T2, i.e., tau 441 protein, was used as the second antibody, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody in Example 6. The results are shown in Table 3.

[0114] (Example 7) As in Example 5, except that an intermediate region recognition tau antibody that recognizes the amino acid sequence from the 185th to the 191st of antibody T3, i.e., tau 441 protein, was used as the second antibody, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody in Example 7. The results are shown in Table 3.

[0115] (Example 8) As in Example 5, except that an intermediate region recognizing tau antibody that recognizes the amino acid sequence from the 218th to the 225th of antibody T4, that is, tau441 protein, was used as the second antibody, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody of Example 8. The results are shown in Table 3.

[0116] Also, with the luminescence intensity which is the detection result of Example 8 as the vertical axis and the luminescence intensity which is the detection result of Reference Example 4 described later as the horizontal axis, the detection results of Example 8 and Reference Example 4 were plotted and compared. The results are shown in FIG. 5.

[0117] (Comparative Example 3) As in Example 5, except that a comparative antibody that recognizes antibody T5, that is, the amino acid sequence from the 395th to the 417th of tau441 protein, was used as the second antibody, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody of this Comparative Example 3. The results are shown in Table 2.

[0118] (Comparative Example 4) As in Example 5, except that a comparative antibody that recognizes antibody T6, that is, the amino acid sequence from the 428th to the 441st of tau441 protein, was used as the second antibody, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area were evaluated for the combination of the first antibody and the second antibody of this Comparative Example 4. The results are shown in Table 2.

[0119] (Reference Example 4) As in Example 8, except that the exosome extract described above was used instead of plasma as the specimen, the luminescence amount was measured for 20 specimens (plasma) as described above to detect phosphorylated tau protein.

[0120] With the luminescence intensity which is the detection result of Example 8 as the vertical axis and the luminescence intensity which is the detection result of Reference Example 4 as the horizontal axis, the detection results of Example 8 and Reference Example 4 were plotted and compared. The results are shown in FIG. 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, with the tau protein detection method according to the present disclosure, tau protein can be detected with good detection accuracy. Similarly, as is clear from the results of Examples 5 to 8 and Comparative Examples 3 and 4, with the tau protein detection method according to the present disclosure, phosphorylated tau protein can be detected with good detection accuracy.

[0123] Also, as shown in FIGS. 2 to 5, in any of Example 2, Example 3, Comparative Example 1, and Example 8, a significant correlation was found with Reference Examples 1 to 4 which are the results using exosome extracts (the straight lines in the figures). Therefore, in the detection method according to the present disclosure using a blood sample such as plasma as a sample, it is judged that the detection results from exosome extracts are well reflected. Therefore, according to the present disclosure, the possibility of loss of tau protein during the exosome extraction process can be effectively avoided, and tau protein or phosphorylated tau protein can be detected well.

[0124] Note that the reference examples using exosome extracts were carried out to correspond to all of Examples 1 to 8 and Comparative Examples 1 to 4. However, in the detailed description of the present invention, reference examples are given only for representative examples or comparative examples to illustrate the comparative relationship (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 convenience of explanation, the comparison results are not shown for all examples and comparative examples, but a significant correlation has been found between all examples and comparative examples and the reference examples. In Comparative Examples 1 to 4, since the secondary antibody recognizes the amino acid sequence of the C-terminal region, although the detection accuracy is inferior to that of Examples 1 to 8, it can be seen that it is the same as Examples 1 to 8 in that the detection results from the exosome extract are well reflected.

[0126] Thus, the tau protein detection method according to the present disclosure is a tau protein detection method for detecting a tau protein or a phosphorylated tau protein from a specimen collected from a subject by an antigen-antibody reaction using two types of antibodies that specifically bind to the tau protein or the phosphorylated tau protein, 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 labeled with a molecule capable of binding to the carrier, 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 between them, the epitopes recognized by the first antibody and the second antibody are amino acid sequences included in the intermediate region or amino acid sequences included 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 such a configuration, tau protein or phosphorylated tau protein in a sample is detected by a sandwich method combining a substantially immobilized first antibody and a labeled second antibody that is not immobilized. At this time, as the first antibody and the second antibody, an antibody having an epitope in the middle region or the N-terminal region is used instead of an antibody having an epitope in the C-terminal region of the tau protein, and a solid-phase carrier for immobilizing the first antibody (a capture carrier on which an antibody as a capture molecule is immobilized) is used. Separately, a non-solid-phase carrier that does not immobilize an antibody (a non-capture carrier that does not immobilize an antibody as a capture molecule) is not used in combination.

[0128] Thereby, even without performing pretreatment such as exosome extraction, tau protein or phosphorylated tau protein contained in exosomes of a blood sample can be detected well. Therefore, the possibility of loss of tau protein during the exosome extraction process can be effectively avoided, and tau protein (or phosphorylated tau protein) can be detected with better sensitivity. In addition, not only is the process of extracting exosomes no longer necessary, but also since a non-solid-phase carrier is not used in combination, there is no need to prepare a non-solid-phase carrier or adjust the abundance ratio of the non-solid-phase carrier to the solid-phase carrier. Therefore, the complication of detecting tau protein can be effectively suppressed or avoided. Furthermore, since an antibody that binds to the C-terminal region does not need to be used as the first antibody and the second antibody, the degree of freedom in selecting two types of antibodies can be improved.

[0129] Note that the present invention is not limited to the description of the above embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments and a plurality of modified examples are also included in the technical scope of the present invention.

[0130] Also, from the above description, many improvements and other embodiments of the present invention will be apparent to those skilled in the art. Therefore, the above description should be construed as illustrative only and provided for the purpose of teaching those skilled in the art the best mode of carrying out the present invention. Without departing from the spirit of the present invention, the details of its structure and / or function can be substantially changed.

Industrial Applicability

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

Claims

1. A tau protein detection method for use in a method for identifying a neurodegenerative disease by detecting tau protein or phosphorylated tau protein from a sample collected from a subject through an 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 capable of binding 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 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 method is characterized in that, without using a non-solid-phase carrier (non-capture carrier) on which no antibody is immobilized, one of the two types of antibodies is first subjected to an antigen-antibody reaction with the specimen, and then the other antibody is subjected to an antigen-antibody reaction with the specimen, thereby forming an immune complex immobilized on the carrier, thereby detecting the tau protein or the phosphorylated tau protein from the specimen. Tau protein detection method.

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 an amino acid sequence from the 103rd to the 371st amino acids of the Tau441 protein. The method for detecting 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 the 149th to the 244th amino acids of the intermediate region. The method for detecting 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 the 188th to the 244th positions of the intermediate region. The method for detecting 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 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 tau protein according to claim 5.

7. the carrier on which the first antibody is immobilized is a magnetic bead, a resin bead, a glass bead, a resin plate, a membrane, or a resin tube; The molecule capable of binding to the carrier is biotin or avidin. A method for detecting 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. A method for detecting 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. A method for detecting 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 Lewy body dementia; The method for detecting tau protein according to claim 9.

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

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