WO2021065306A1 - 血液試料を検体とするタウタンパク質検出方法 - Google Patents

血液試料を検体とするタウタンパク質検出方法 Download PDF

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WO2021065306A1
WO2021065306A1 PCT/JP2020/033211 JP2020033211W WO2021065306A1 WO 2021065306 A1 WO2021065306 A1 WO 2021065306A1 JP 2020033211 W JP2020033211 W JP 2020033211W WO 2021065306 A1 WO2021065306 A1 WO 2021065306A1
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antibody
tau protein
tau
amino acid
protein
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剛章 香束
優花 佐野
利明 馬場
吉田　博
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Nipro Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • the present invention relates to a tau protein detection method using a blood sample as a sample, and more particularly to a tau protein detection method capable of satisfactorily detecting tau protein contained in an exosome in a blood sample.
  • 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 and stabilizes microtubules.
  • MAP microtubule-associated protein
  • Tau protein is known as a phosphorylated protein in which multiple serines or threonins in the amino acid sequence are phosphorylated. When this phosphorylation is abnormal, Alzheimer's disease (AD) and frontotemporal dementia It is thought to be involved in neurodegenerative diseases such as (FTD) and Levy body dementia. Therefore, it has been conventionally studied to use tau protein and phosphorylated tau protein as biomarkers for neurodegenerative diseases.
  • Patent Document 1 As a specific method for detecting tau protein or phosphorylated tau protein, for example, the method disclosed in Patent Document 1 is known.
  • molecules other than tau protein and phosphorylated tau protein are listed as biomarkers, and samples for detecting these biomarkers include blood (whole blood), serum, plasma, urine, and interstitial fluid.
  • Biomarkers such as fluid, ascites, cervical swab, tears, plasma, cheek swab, skin, brain tissue, cerebrospinal fluid, etc. are listed.
  • vesicles such as exosomes (exosomes), fine particles, microvesicles, nanosomes, extracellular vesicles, and ectosomes are isolated from these biological samples, or these vesicles in the biological sample are concentrated in this manner.
  • One or more biomarkers have been detected in vesicles. If the biomarker is phosphorylated tau protein, the phosphorylated tau protein is detected using a composition containing an antibody.
  • Patent Document 2 in the presence of non-capture beads, an immune complex of a phosphorylated tau protein in a biological sample, a capture antibody, and a detection antibody is formed on the capture beads to form an immune complex.
  • a method for detecting a signal derived from is disclosed.
  • non-capture beads in which the epitope of the capture antibody and the epitope of the detection antibody are different and do not form an immune complex are allowed to coexist so as to be 1.5 or more with respect to the capture bead 1. ing.
  • Patent Document 2 by forming an immune complex by coexisting non-capture beads so as to be 1.5 times or more as many as the capture beads, the sensitivity is such that the Alzheimer's disease (AD) patient and the target patient can be distinguished. Phosphorylated tau protein can be measured (detected). However, there is no specific reason why phosphorylated tau protein can be detected (measured) satisfactorily due to the presence of non-captured beads.
  • the present inventors extracted and crushed exosomes from a blood sample, and when the amount of exosome protein was confirmed by electrophoresis, it was possible to confirm the amount of tau protein at the nanogram level per 1 mL. Therefore, it was considered that if the tau protein contained in the exosome of the blood sample could be detected with good reactivity, the detection accuracy of the tau protein could be further improved.
  • the conventional method requires a process for extracting vesicles such as exosomes, which complicates the tau protein detection method.
  • the present invention has been made to solve such a problem, and a tau protein or a phosphorylated tau protein can be obtained with better sensitivity by using a blood sample as a sample and avoiding or suppressing complicated detection processing. It is an object of the present invention to provide a detection method capable of detecting.
  • the tau protein detection method according to the present invention was collected from a subject by an antigen-antibody reaction using two types of antibodies that specifically bind to tau protein or phosphorylated tau protein in order to solve the above-mentioned problems.
  • a tau protein detection method for detecting tau protein or phosphorylated tau protein from a sample wherein the sample is a blood sample and one of the two types of the antibodies is used or immobilized on a carrier.
  • a first antibody labeled with a molecule capable of binding to a carrier, the other is a second antibody labeled without being immobilized on the carrier, the tau protein in the N-terminal region, C-terminal region, and these.
  • the epitopes recognized by the first antibody and the second antibody, respectively, when divided into the intermediate regions located between them are the amino acid sequence contained in the intermediate region or the amino acid sequence contained in the N-terminal region.
  • the configuration is such that 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.
  • tau protein or phosphorylated tau protein in a sample is detected by a sandwich method in which a substantially immobilized first antibody and a non-immobilized labeled second antibody are combined.
  • a substantially immobilized first antibody and a non-immobilized labeled second antibody are combined.
  • the first antibody and the second antibody an antibody having an epitope in the C-terminal region of the tau protein is not used, but an antibody having an epitope in the intermediate region or the N-terminal region is used, and the first antibody is immobilized.
  • the immobilized carrier capture carrier on which the antibody that is the capture molecule is immobilized
  • a non-immobilized carrier that does not immobilize the antibody do not use together.
  • the N-terminal region is one of the tau 441 proteins. It is a region consisting of the 43rd to 44th amino acid sequences, and the intermediate region may be a region consisting of the 103rd to 371st amino acid sequences of the tau 441 protein.
  • the epitope when an amino acid sequence serving as an epitope of the antibody is contained in the intermediate region, the epitope is a proline consisting of the 149th to 244th amino acid sequences of the intermediate region.
  • the configuration may be included in the rich region.
  • the epitope when an amino acid sequence serving as an epitope of the antibody is contained in the intermediate region, the epitope is a proline consisting of the 188th to 244th amino acid sequences of the intermediate region.
  • the configuration may be included in the rich region.
  • the phosphorylated tau protein is the tau 441 based on the tau 441 protein having the longest amino acid sequence of 441 residues among the tau protein isoforms. 46th, 175th, 181st, 185th, 198th, 199th, 202nd, 205th, 208th, 210th, 212th, 214th, 217th, 231st, 235th, 237th, At least one of the 238th, 262nd, and 356th amino acid residues may be phosphorylated.
  • the phosphorylated tau protein serving as the antigen of the first antibody is the 175th, 181st, 185th, 198th, 199th, 202th, 205th of the tau 441 protein.
  • the composition may be such that at least one of the amino acid residues at positions 208, 210, 212, 214, 217, 231 and 235, 237 or 238 is phosphorylated. ..
  • 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, and is bound to the carrier. It may be configured such that the possible molecule is biotin or avidin.
  • the label of the second antibody may have a structure of an enzyme, a fluorescent dye, a fluorescent protein, or biotin.
  • the sample may be a blood sample obtained from a subject suspected of having a neurodegenerative disease.
  • the neurodegenerative disease may be at least one of Alzheimer's disease (AD), frontotemporal dementia (FTD), and Lewy body dementias. Good.
  • AD Alzheimer's disease
  • FTD frontotemporal dementia
  • Lewy body dementias Lewy body dementias. Good.
  • the present invention may 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 having the above constitution.
  • the present invention provides a detection method capable of detecting tau protein or phosphorylated tau protein with better sensitivity by avoiding or suppressing complicated detection processing using a blood sample as a sample with the above configuration. It has the effect of being able to do it.
  • tau protein detection method two types of antibodies (anti-tau antibody or anti-phosphorylated tau antibody) that specifically bind to tau protein or phosphorylated tau protein are used, and the tau protein is collected from a subject by an antigen-antibody reaction.
  • This is a method for detecting tau protein or phosphorylated tau protein from a sample.
  • a blood sample was used as the sample, and it was used as an anti-tau antibody or an anti-phosphorylated tau antibody by being immobilized on a carrier, or labeled with a molecule capable of binding to the carrier (substantially immobilized).
  • a first antibody and a second antibody that is labeled and used without being immobilized on a carrier.
  • a first antibody and a second antibody that is labeled and used without being immobilized on a carrier.
  • an anti-tau antibody that specifically binds to tau protein and an anti-phosphorylated tau antibody that specifically binds to phosphorylated tau protein are collectively abbreviated as "tau antibody”. ..
  • the epitopes recognized by the first antibody and the second antibody, respectively are amino acid sequences contained in the intermediate region.
  • an amino acid sequence contained in the N-terminal region that is, as the first antibody, a tau antibody having an amino acid sequence contained in the intermediate region of the tau protein or the phosphorylated tau protein as an epitope may be used, or it may be contained in the N-terminal region of the tau protein or the phosphorylated tau protein.
  • a tau antibody having the amino acid sequence as an epitope may be used as the first antibody.
  • a tau antibody having an amino acid sequence contained in the N-terminal region of the tau protein or the phosphorylated tau protein as an epitope may be used, or in the intermediate region of the tau protein or the phosphorylated tau protein.
  • a tau antibody using the contained amino acid sequence as an epitope may be used.
  • one of the two types of tau antibodies may first undergo an antigen-antibody reaction with a blood sample, and then the other may undergo an antigen-antibody reaction with a blood sample.
  • a first reaction solution was prepared by mixing a second antibody that was not previously immobilized with a blood sample and reacting with an antigen-antibody, and then immobilized with the first reaction solution. The first antibody may be reacted with an antigen-antibody reaction.
  • a first reaction solution is prepared by subjecting a blood sample to an antigen-antibody reaction with a first antibody immobilized first, and then a second antibody not immobilized with the first reaction solution is prepared. And may be reacted with an antigen-antibody.
  • 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 Reference 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 2: International Publication No. 2013/180238, etc.
  • Phosphorylated tau protein is a product in which some of the amino acids that make up tau protein are phosphorylated.
  • tau protein As described in Reference 1 or Reference 2, six types of tau protein are typically known as isoforms.
  • the MAPT gene which encodes tau protein, has 16 exons, and exons 1 to 13 are expressed in the central nervous system. Of these, alternative splicing occurs for exons 2, 3 and 10, and 6 types of isoforms are produced as described above.
  • alternative splicing of exon 2 or exon 3 of the MAPT gene results in 0 to 2 repetitive sequences N (0N to 2N) on the N-terminal side, and exons of the MAPT gene.
  • Alternative splicing of 10 may result in 3 (1R-3R) or 4 (1R-4R) repetitive sequences R in the microtube binding region.
  • the six isoforms of tau protein are 0N3R (352 amino acid residues), 1N3R (381 amino acid residues), 2N3R (410 amino acid residues), It can be expressed as 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 types of isoforms.
  • tau protein 2N4R which is an isoform in which alternative splicing does not occur in any of exons 2, 3 and 10, that is, tau protein 2N4R having the longest amino acid sequence of 441 residues among the 6 types of isoforms.
  • the ordinal number of the amino acid sequence is specified.
  • tau protein 2N4R is referred to as "tau 441 protein" based on the number of amino acid residues.
  • the tau 441 protein (2N4R) is described in Reference 1, and as shown in FIG. 1, an N-terminal region, a proline-rich domain (PRD), and a microtubule-binding domain (MBD). ), C-terminal region can be divided into four regions.
  • the N-terminal region is composed of the 1st to 148th amino acid sequences (aa1-148)
  • the proline-rich region is composed of the 149th to 244th amino acid sequences (aa149-244)
  • the microtubule binding region is 245.
  • the C-terminal region is composed of the amino acid sequence from the 373rd to the 441th (aa373-441).
  • the two repetitive sequences N on the N-terminal side are described as “N1” and “N2”, respectively, and the four repetitive sequences R of the microtubule binding region are "R1", “R2", and “R3”, respectively. , "R4".
  • the amino acid sequence of the tau protein recognized by the tau antibody is contained in at least the intermediate region, and further contained in the N-terminal region in addition to the intermediate region.
  • the intermediate region of the tau protein according to the present disclosure may be a region located between the N-terminal region and the C-terminal region, and therefore, a region including a proline-rich region and a microtubule binding region, that is, The region composed of the amino acid sequences (aa149-372) from the 149th to the 372nd position can be used as an intermediate region.
  • the N-terminal region is defined as a wide region before the proline-rich region, but the narrow region from the N-terminal to the front of the repetitive sequence N is defined as the "narrowly defined” N-terminal region. be able to.
  • the tau protein isoform contains 0N3R and 0N4R, which do not contain the repetitive sequence N. That is, if the amino acid sequence recognized by the tau antibody is contained in the N-terminal region in the "narrow sense", the isoforms of 0N3R and 0N4R can be detected well.
  • the "narrowly defined" N-terminal region is composed of the 1st to 44th amino acid sequences (aa1-44).
  • the intermediate region located between the N-terminal region and the C-terminal region can be defined as a region extending toward the N-terminal side.
  • the reference N-terminal region is composed of the amino acid sequences (aa1-148) from the 1st to the 148th, but as described above, the N-terminal region in the "narrow sense" is from the 1st to the 44th. It is composed of an amino acid sequence (aa1-44). Therefore, the "broadly defined” intermediate region can be expanded to the 45th amino acid residue on the N-terminal side.
  • the repetitive sequence N may not be included due to alternative splicing. Therefore, when the tau 441 protein is used as a reference, the amino acid residue next to the end point of the repetitive sequence N is set as the start point of the intermediate region. It should be. Since the end point of the repetitive sequence N is the 102nd amino acid residue of the tau 441 protein, the start point of the intermediate region in the "broad sense" may be the 103rd amino acid residue. Therefore, in the present embodiment, the "broadly defined" intermediate region is preferably composed of the amino acid sequences from positions 103 to 372 (aa103-372).
  • intermediate region in the "broad sense” and the N-terminal region in the “narrow sense” in the present disclosure are defined not based on the ordinal number of the amino acid sequence of the tau 441 protein, but on the repeat sequence N and the repeat sequence R. Can also be expressed.
  • the end point of the amino acid sequence encoded by the repetitive sequence N may be used as the starting point of the intermediate region in the "broad sense", or the exon 2 and exon 3 may be selectively selected.
  • the position where these exon-encoded amino acid sequences are deleted by splicing may be used as the starting point of the "broadly defined” intermediate region.
  • the end point of the intermediate region in the "broad sense” may be the end point of the amino acid sequence encoded by the repetitive sequence R, that is, exons 9 to 12 of the MAPT gene, as the end point of the intermediate region in the "broad sense".
  • the "narrowly defined" N-terminal region is the region from the N-terminal to the starting point of the intermediate region, that is, from the N-terminal to the starting point of the amino acid sequence encoded by exon 2 or exon 3 of the MAPT gene. Can be expressed.
  • the phosphorylated tau protein in the present disclosure may be one in which at least one amino acid residue of the tau protein including the above-mentioned six isoforms is phosphorylated.
  • at least one of the 235th, 237th, 238th, 262nd, and 356th amino acids is phosphorylated. These amino acid residues are either serine or threonine.
  • typical phosphorylated tau proteins are 175th, 181st, 185th, 198th, 199th, 202th, 205th, 208th, 210th, 212th, 214th, 217th, At least one of the 231st, 235th, 237th or 238th amino acid residues is phosphorylated. These amino acid residues are contained in the proline-rich region (aa149-244). Therefore, the anti-phosphorylated tau antibody that makes it possible to recognize the phosphorylation of these amino acid residues corresponds to the first antibody that recognizes the intermediate region. For example, in the examples described later, as an example of the anti-phosphorylated tau antibody (first antibody), the one in which the 181st amino acid residue (threonine) is phosphorylated is used.
  • the tau protein and phosphorylated tau protein in the present disclosure are not limited to the above-mentioned six types of isoforms, and may be different isoforms (for example, unknown isoforms).
  • 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., if necessary) from the MAPT gene, and is limited to known isoforms. It is not something that is done.
  • the tau antibody used in the tau protein detection method according to the present disclosure is an antibody using the above-mentioned tau protein or phosphorylated tau protein as an antigen.
  • the tau antibody as described above, it is used by being immobilized on a carrier, or is used by being labeled without being immobilized on a carrier with a first antibody labeled with a molecule capable of binding to the carrier. Two types of the second antibody are used in combination.
  • the carrier on which the first antibody is immobilized is not particularly limited, and those known in the field of antigen-antibody reaction can be preferably used. Specific examples thereof include magnetic beads, resin beads, glass beads, resin plates, membranes, resin tubes and the like.
  • the type of resin material (polymer compound) used for the resin beads, the resin plate, the resin tube, and the like is not particularly limited, and a resin material known for the antigen-antibody reaction can be preferably used.
  • the specific configuration of the magnetic beads is not particularly limited, and examples thereof include a configuration in which the surface of a particulate magnetic material is coated with a resin material.
  • the material used for the membrane is not particularly limited, and a known material such as nitrocerus or polyvinylidene fluoride (PVDF) can be publicly used.
  • the specific configuration (shape, size, etc.) of the beads, plate, membrane, or tube is not particularly limited, and any known configuration that is judged to be suitable for the tau protein detection method may be used.
  • the method of immobilizing (immobilizing) the first antibody on the carrier is not particularly limited, and a method known in the field of antigen-antibody reaction can be preferably used. Alternatively, a commercially available antibody immobilization kit may be used to immobilize the first antibody on a suitable carrier in a timely manner.
  • the first antibody may be an antibody labeled with a molecule capable of binding to the carrier, instead of being directly immobilized on the carrier.
  • the first antibody may be labeled with a labeled molecule such as biotin or avidin, and the first antibody may be immobilized on the carrier by binding to the carrier. Therefore, the first antibody in the present embodiment includes not only an antibody that is directly immobilized on the carrier but also an antibody that is indirectly immobilized via a molecule that can bind to the carrier. Therefore, in the present embodiment, the first antibody can be described as an antibody substantially immobilized on a carrier.
  • the labeled molecule that can bind to the carrier is not limited to the above-mentioned biotin or avidin, and various known molecules can be used.
  • the label applied to the second antibody is not particularly limited, and those known in the field of antigen-antibody reaction can be preferably used. Specific examples thereof include enzymes, fluorescent dyes, fluorescent proteins, biotin and the like.
  • the method for labeling the second antibody is not particularly limited, and a method known in the field of antigen-antibody reaction can be preferably used. Alternatively, a commercially available antibody labeling kit may be used to timely label the second antibody with a suitable type of labeling.
  • the epitope recognized by the first antibody and the second antibody may be an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein, or an amino acid sequence contained in the N-terminal region. That is, the first antibody and the second antibody may not be tau antibodies having an amino acid sequence contained in the C-terminal region of tau protein or phosphorylated tau protein as an epitope.
  • a tau antibody having an amino acid sequence contained in the intermediate region of the tau protein or the phosphorylated tau protein as an epitope may be used, or is contained in the N-terminal region of the tau protein or the phosphorylated tau protein.
  • a tau antibody having the amino acid sequence as an epitope may be used.
  • a tau antibody having an amino acid sequence contained in the N-terminal region of the tau protein or the phosphorylated tau protein as an epitope may be used, or in the intermediate region of the tau protein or the phosphorylated tau protein.
  • a tau antibody using the contained amino acid sequence as an epitope may be used.
  • the first antibody (1) an antibody having an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein as an epitope is used, and as the second antibody, (2) tau.
  • 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).
  • the first antibody may be a tau antibody having an amino acid sequence contained in the N-terminal region as an epitope.
  • a method for producing these first antibody and second antibody that is, an antibody having an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein as an epitope, and N of tau protein or phosphorylated tau protein.
  • the method for producing an antibody using the amino acid sequence contained in the terminal region as an epitope is not particularly limited, and it may be produced by a known method using tau protein or phosphorylated tau protein as an antigen.
  • a tau antibody having an amino acid sequence contained in the intermediate region as an epitope is referred to as an "intermediate region recognition tau antibody", and a tau antibody having an amino acid sequence contained in the N-terminal region as an epitope is referred to as "N-terminal region recognition”.
  • tau antibody a tau antibody having an amino acid sequence contained in the C-terminal region as an epitope, which is not used as the first antibody or the second antibody in the present disclosure, may also be referred to as "C-terminal region recognition tau antibody" for convenience of explanation.
  • the tau protein or phosphorylated tau protein to be an antigen may be a commercially available one, or may be prepared by a known method using a gene recombination technique.
  • the host (host) cell expressing tau protein or phosphorylated tau protein may be a bacterium (prokaryotic cell) such as Escherichia coli, or a eukaryotic cell such as cultured cells of yeast or animals and plants. Alternatively, a cell-free protein synthesis system may be used.
  • a hybridoma of mouse spleen is prepared by immunizing a mouse with a binding protein in which an antigen tau protein or a phosphorylated tau protein is covalently bound to KLH (Keyhole Limpet Hemocyanin), and this hybridoma is produced. Cultivate and extract tau antibody from the supernatant of the culture broth. The recognition site of the obtained tau antibody may be identified using a peptide having each corresponding amino acid sequence.
  • the tau antibody corresponding to the intermediate region recognition tau antibody and the N-terminal region recognition tau antibody is commercially available, In either case, a commercially available antibody may be used.
  • a commercially available antibody may be used.
  • any of these tau antibodies is commercially available and one is not commercially available (eg, the intermediate region recognition tau antibody is commercially available and the N-terminal region recognition tau antibody is not commercially available, or the first antibody.
  • the intermediate region recognition tau antibody used as the second antibody is commercially available and the intermediate region recognition tau antibody used as the second antibody is not commercially available
  • the commercially available antibody and the tau antibody prepared as described above may be used in combination. Good.
  • the intermediate region recognition tau antibody used as the first antibody or the second antibody in the present disclosure has an amino acid sequence contained in the intermediate region of tau protein or phosphorylated tau protein as an epitope (recognition sequence). Therefore, even if the epitope of the intermediate region recognition tau antibody is an amino acid sequence contained in the intermediate region in the "broad sense" composed of the 45th to 372nd amino acid sequences (aa45-372) based on the tau 441 protein.
  • the amino acid sequence contained in the intermediate region of the reference region composed only of the proline-rich region and the microtubule binding region
  • the amino acid sequence contained in the intermediate region of the reference composed only of the proline-rich region and the microtubule binding region
  • the intermediate region recognition tau antibody is an anti-phosphorylated tau antibody that recognizes phosphorylated tau protein
  • the phosphorylation site of the tau protein recognized by the intermediate region recognition tau protein (position of phosphorylated amino acid residue). ) May be included in the "broadly defined” intermediate region or in the reference intermediate region, as described above.
  • the phosphorylation site recognized by the intermediate region recognition tau antibody may be an amino acid sequence contained in the proline-rich region (amino acid sequence from 149th to 244th, aa149-244).
  • the N-terminal region recognition tau antibody used as the first antibody or the second antibody in the present disclosure uses the amino acid sequence contained in the N-terminal region of tau protein as an epitope. Therefore, the epitope of the N-terminal region recognition tau antibody may be an amino acid sequence contained in the reference N-terminal region composed of the amino acid sequences (aa1-148) from the 1st to the 148th positions, or from the 1st position. It may be an amino acid sequence contained in the "narrowly defined" N-terminal region composed of up to the 44th amino acid sequence (aa1-44).
  • the epitope of the intermediate region recognition tau antibody and the N-terminal region recognition tau antibody has a more preferable range (suitable region) in the intermediate region or the N-terminal region.
  • the amino acid sequence serving as an epitope of the intermediate region recognition tau antibody is contained in the 181st to 191st amino acid sequence (aa181-191) in the intermediate region, or the 218th to 225th amino acids. It is preferably included in the sequence (aa218-225).
  • the amino acid sequence serving as an epitope of the N-terminal region recognition tau antibody is contained in the 1st to 20th amino acid sequences (aa1-20) of the N-terminal region, or the 16th to 24th amino acid sequences. It is preferably contained in (aa16-24). If the intermediate region recognition tau antibody and the N-terminal region recognition tau antibody have an amino acid sequence contained in a suitable region as an epitope, the detection accuracy of tau protein or phosphorylated tau protein can be further improved. ..
  • tau protein detection method In the tau protein detection method according to the present disclosure, a blood sample is used as a sample, and the above-mentioned two types of the first antibody and the second antibody are reacted with this sample to obtain tau protein or phosphorylated tau protein in the sample. Is detected.
  • the tau antibody having the C-terminal region of the tau protein or the phosphorylated tau protein as an epitope is not used, and the intermediate region or the N-terminal region is used.
  • a non-immobilized carrier that does not immobilize an antibody (a capture carrier that immobilizes an antibody that is a capture molecule) while using an antibody as an epitope and immobilizes the first antibody (a capture carrier that immobilizes an antibody that is a capture molecule).
  • a non-capture carrier that does not immobilized the antibody that is a capture molecule) is not used in combination. Thereby, tau protein or phosphorylated tau protein contained in the exosome of the blood sample can be satisfactorily detected.
  • a commercially available antibody that recognizes the 159th to 163rd amino acid sequences (aa159-163) of tau protein is used as a “capture antibody”, and the 181st amino acid (threonine) of phosphorylated tau protein is used.
  • a commercially available antibody that recognizes phosphorylation of residues is used as a "detection antibody”.
  • the capture antibody is bound to the capture beads, and the capture antibody is first bound to the target protein (phosphorylated tau protein).
  • the detection antibody is further bound to the target protein bound to the capture antibody to form an immune complex of capture antibody-target protein-detection antibody.
  • the capture antibody recognizes the amino acid sequence of aa159-163 of tau protein when used in a state of being bound to capture beads, it corresponds to the first antibody and intermediate region recognition tau antibody in the present disclosure.
  • the detection antibody is a biotin-labeled antibody that is not bound to capture beads and recognizes the phosphorylation of the 181st amino acid residue of the phosphorylated tau protein. Corresponds to the intermediate region recognition tau antibody.
  • 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 obtained. It is reacted with phosphorylated tau protein.
  • the reason is unknown, but a large amount of non-capturing beads (non-immobilized carrier that does not immobilize the antibody) are used in combination. It seems necessary.
  • the tau protein detection method according to the present disclosure As is clear from the comparison results of Examples and Reference Examples described later, the detection results of tau protein or phosphorylated tau protein detected from exosomes extracted from blood samples. There is a significant correlation between the detection result of tau protein or phosphorylated tau protein detected from the blood sample itself (FIGS. 2 to 5). Further, as is clear from the results of Examples and Comparative Examples, the tau protein detection method according to the present disclosure has a sensitivity of 70% or more, a specificity of 70% or more, and a ROC curve (Receiver Operating Characteristic curve). The area of the receiver operating characteristic curve) is 0.70 or more, which is an extremely excellent detection accuracy.
  • the antibody in addition to not using a tau antibody having the C-terminal region as an epitope, the antibody is immobilized by using only an immobilization carrier that immobilizes the first antibody.
  • a non-immobilized carrier non-capturing carrier
  • tau protein detection method it is possible to detect tau protein or phosphorylated tau protein contained in exosomes by using a blood sample which is a sample before exosome extraction without extracting exosomes. Is. Therefore, the possibility of loss of tau protein in the process of exosome extraction can be effectively avoided, and tau protein (or phosphorylated tau protein) can be detected with better sensitivity, and the detection of tau protein is complicated. It can be effectively suppressed or avoided.
  • 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.
  • Specific blood samples include whole blood, plasma or serum. Of these, plasma is more preferred.
  • the method for separating plasma from whole blood is not particularly limited, and the method separated from whole blood collected by a known blood collection method using an anticoagulant such as heparin salt, citrate, ethylenediaminetetraacetic acid (EDTA) salt is used. It may be used.
  • a tau antibody in which the recognition sequence (epithodate) of the tau protein or the phosphorylated tau protein is optimized is used, and these tau antibodies are substantially immobilized on the carrier. If it is a method in which the first antibody and the labeled second antibody are used in combination and the first antibody and the second antibody are reacted with the blood sample as a sample, other specific treatments thereof.
  • the processing conditions and the order of the processing are not particularly limited.
  • the blood sample as a sample is appropriately diluted and reacted with the second antibody first, and then the first antibody is reacted, and then the carrier is separated and washed.
  • the reaction sequence of the first antibody and the second antibody, the dilution of the blood sample, the separation of the carrier, the washing of the immune complex, and other specific methods, conditions, reagents used, and the like are not particularly limited. If tau protein or phosphorylated tau protein can be detected well, the reaction order of the first antibody and the second antibody may be appropriately selected, and some treatments such as dilution, separation, and washing may be omitted. However, treatments other than these treatments may be performed, the order of these treatments may be changed, the same treatment or different reagents may be used, or the same treatment may be repeated a plurality of times.
  • tau protein or phosphorylated tau protein is detected by adding a luminescent substrate to the washed carrier and reacting it, and measuring the amount of luminescence (emission intensity) thereof with a commercially available photometer.
  • the detection of tau protein or phosphorylated tau protein is not limited to such a method by measuring the amount of luminescence, and other known methods may be used.
  • tau protein or phosphorylated tau protein may be detected using a known detection kit.
  • the tau protein detection method according to the present disclosure can satisfactorily detect tau protein or phosphorylated tau protein, it can be suitably used for the purpose of identifying (differentiating or diagnosing) neurodegenerative diseases. That is, in the present disclosure, a human (subject) who is a sample collector (subject) by detecting tau protein or phosphorylated tau protein in a blood sample as a sample by using the above-mentioned tau protein detection method. Or mammals) may include methods of identifying neurodegenerative diseases.
  • 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 judges not only those who have developed neurodegenerative disease and those who have symptoms suspected of developing it, but also those who have no symptoms but may have neurodegenerative disease. It may be a person who wants to do so, that is, a healthy person.
  • the neurodegenerative disease to be identified in the tau protein detection method according to the present disclosure is a disease that develops (or may develop, or is suggested to develop) due to an abnormality in tau protein. All you need is. Diseases that are thought to be mainly due to abnormal accumulation of phosphorylated tau protein are collectively called "tauopathy".
  • the tau protein detection method according to the present disclosure can be suitably used for identifying such tauopathy.
  • Specific neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), frontotemporal dementia (FTD), Lewy body dementias, and the like.
  • tau antibody Antibodies, samples, reagents, etc., and detection methods
  • 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 described above.
  • the tau protein or phosphorylated tau protein serving as an 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 each corresponding amino acid sequence.
  • sample As samples of Examples and Comparative Examples, a total of 20 types of plasma, including 10 types of plasma provided by 10 AD patients and 10 types of plasma provided by 10 healthy subjects, were used. In addition, in the reference example, 20 types of exosomes extracted from these plasmas by a polymer precipitation method (exosome extracts) were used.
  • a 2.0% bovine serum albumin-containing phosphate buffer was used as the diluent for diluting the sample.
  • a phosphate buffer solution containing 0.05% Tween 20 was used as a cleaning solution.
  • the product name Lumigen APS-5 manufactured by Fuji Film Wako Pure Chemical Industries, Ltd. was used.
  • a carrier for immobilizing the first antibody a product name dynabead magnetic particles (magnetic beads) manufactured by Thermo was used.
  • a method for immobilizing the first antibody on the magnetic particles the method described in the dynabead package insert was used.
  • ALP Purified calf intestinal alkaline phosphatase manufactured by Thermo was used as a label for the second antibody.
  • ALP Purified calf intestinal alkaline phosphatase
  • Alkaline Phosphatase Labeling Kit of Dojin Chemical Products was used, and the second antibody was labeled by the method described in the package insert.
  • a diluted sample was prepared by mixing 1 part by volume of human plasma (Example or Comparative Example) or exosome extract (reference example) as a sample and 1 part by volume of a diluent. To 2 parts by volume of this diluted sample, 2 parts by volume of the diluted solution and 4 parts by volume of the ALP-labeled antibody solution as the second antibody were further added and mixed. As a result, the first reaction solution was prepared and reacted at 37 ° C. for a predetermined time.
  • Magnetic particles were separated from the second reaction solution after the reaction and washed with a cleaning solution. Eight parts by volume of a luminescent substrate was added to the washed magnetic particles and reacted at 25 ° C. for several seconds to ten and several seconds, and the emission intensity (emission amount) was measured to detect tau protein or phosphorylated tau protein.
  • the sensitivity and specificity of each Example, Comparative Example, or Reference Example were calculated and an ROC curve was created, and the ROC curve was calculated.
  • the area (ROC area) was calculated.
  • the graph of the comparison result is only shown, and the description of the result such as sensitivity is omitted.
  • Example 1 As the first antibody, an intermediate region recognition tau antibody that recognizes the 218th to 225th amino acid sequence (aa218-225) of antibody T4, that is, tau 441 protein is used, and as the second antibody, antibody T1, that is, 1 of tau 441 protein.
  • an N-terminal region-recognizing tau antibody that recognizes the 20th to 20th amino acid sequence (aa1-20) tau protein was detected by measuring the amount of luminescence of 20 types of samples (plasma) as described above.
  • Example 2 About 20 kinds of samples (plasma) in the same manner as in Example 1 except that the N-terminal region recognition tau antibody that recognizes the 16th to 24th amino acid sequences of antibody T2, that is, the tau 441 protein was used as the second antibody. As described above, the amount of luminescence was measured 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 of Example 2 were evaluated. The results are shown in Table 2.
  • Example 2 and Reference Example 1 are plotted with the emission intensity which is the detection result of this Example 2 as the vertical axis and the emission intensity which is the detection result of Reference Example 1 described later as the horizontal axis. Contrast. The result is shown in FIG.
  • Example 3 As the second antibody, 20 kinds of samples (plasma) were described in the same manner as in Example 1 except that an intermediate region recognition tau antibody that recognizes the 185th to 191st amino acid sequences of antibody T3, that is, tau 441 protein was used. As shown, tau protein was detected by measuring the amount of luminescence. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Example 3 were evaluated. The results are shown in Table 2.
  • Example 3 and Reference Example 2 are plotted with the emission intensity which is the detection result of this Example 3 as the vertical axis and the emission intensity which is the detection result of Reference Example 2 described later as the horizontal axis. Contrast. The result is shown in FIG.
  • Example 4 As the second antibody, the amount of luminescence was measured for 20 types of samples (plasma) as described above in the same manner as in Example 1 except that antibody T4, that is, the same antibody as the first antibody was used as the intermediate region recognition tau antibody. Tau protein was detected. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Example 4 were evaluated. The results are shown in Table 2.
  • Comparative Example 1 As the second antibody, 20 kinds of samples (plasma) emit light as described above in the same manner as in Example 1 except that a comparative antibody that recognizes the 395th to 417th amino acid sequences of antibody T5, that is, tau 441 protein was used. The amount was detected and tau protein was measured. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Comparative Example 1 were evaluated. The results are shown in Table 2.
  • Comparative Example 2 As the second antibody, 20 kinds of samples (plasma) emit light as described above in the same manner as in Example 1 except that a comparative antibody that recognizes the 428th to 441st amino acid sequences of antibody T6, that is, tau 441 protein was used. The amount was measured and tau protein was detected. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Comparative Example 2 were evaluated. The results are shown in Table 2.
  • Example 2 and Reference Example 1 were plotted and compared with the emission intensity of the detection result of Example 2 on the vertical axis and the emission intensity of the detection result of Reference Example 1 on the horizontal axis. The result is shown in FIG.
  • Example 3 and Reference Example 2 were plotted and compared with the emission intensity of the detection result of Example 3 on the vertical axis and the emission intensity of the detection result of Reference Example 2 on the horizontal axis. The result is shown in FIG.
  • the antibody Tp that is, the phosphorylated tau protein (pT181) in which the 181st amino acid (threonine) of the tau 441 protein is phosphorylated is used as an antigen (that is, the phosphorylation site existing in the intermediate region is recognized).
  • a region-recognition tau antibody is used, and as a second antibody, an N-terminal region-recognition tau antibody that recognizes the 1st to 20th amino acid sequences of antibody T1, that is, tau 441 protein is used, and 20 types of samples (plasma) are described above.
  • Phosphorescent tau protein was detected by measuring the amount of luminescence.
  • Example 6 About 20 kinds of samples (plasma) in the same manner as in Example 5 except that the N-terminal region recognition tau antibody that recognizes the 16th to 24th amino acid sequences of antibody T2, that is, the tau 441 protein was used as the second antibody. As described above, the amount of luminescence was measured to detect phosphorylated tau protein. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Example 6 were evaluated. The results are shown in Table 3.
  • Example 7 As the second antibody, 20 kinds of samples (plasma) were described in the same manner as in Example 5 except that the antibody T3, that is, the intermediate region recognition tau antibody that recognizes the amino acid sequences 185 to 191 of the tau 441 protein was used. Phosphorylated tau protein was detected by measuring the amount of luminescence as shown. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Example 7 were evaluated. The results are shown in Table 3.
  • Example 8 As the second antibody, 20 kinds of samples (plasma) were described in the same manner as in Example 5 except that an intermediate region recognition tau antibody that recognizes the 218th to 225th amino acid sequences of antibody T4, that is, tau 441 protein was used. Phosphorylated tau protein was detected by measuring the amount of luminescence as shown. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Example 8 were evaluated. The results are shown in Table 3.
  • Examples 8 and 4 are plotted with the emission intensity which is the detection result of this Example 8 as the vertical axis and the emission intensity which is the detection result of Reference Example 4 described later as the horizontal axis. Contrast. The result is shown in FIG.
  • Example 3 As the second antibody, 20 kinds of samples (plasma) emit light as described above in the same manner as in Example 5 except that a comparative antibody that recognizes the 395th to 417th amino acid sequences of antibody T5, that is, tau 441 protein was used. Phosphorylated tau protein was detected by measuring the amount. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Comparative Example 3 were evaluated. The results are shown in Table 2.
  • Example 4 As the second antibody, 20 kinds of samples (plasma) emit light as described above in the same manner as in Example 5 except that a comparative antibody that recognizes the amino acid sequences 428 to 441 of the antibody T6, that is, the tau 441 protein was used. Phosphorylated tau protein was detected by measuring the amount. Based on the results, the sensitivity, specificity, and ROC area of the combination of the first antibody and the second antibody of Comparative Example 4 were evaluated. The results are shown in Table 2.
  • Example 8 and Reference Example 4 were plotted and compared with the emission intensity of the detection result of Example 8 on the vertical axis and the emission intensity of the detection result of Reference Example 4 on the horizontal axis. The result is shown in FIG.
  • the tau protein detection method according to the present disclosure can detect tau protein with good detection accuracy.
  • the tau protein detection method according to the present disclosure can detect phosphorylated tau protein with good detection accuracy.
  • Example 2 is an example of an N-terminal region recognition tau antibody (T2) having an epitope in the N-terminal region
  • Example 3 is an intermediate region
  • Example 8 is an example of an intermediate region recognition tau antibody (T3) having an epitope in
  • Comparative Example 1 is an example of a comparative antibody (T5) having an epitope in the C-terminal region
  • Example 8 is an example having an epitope in the intermediate region.
  • This is an example of an intermediate region recognition tau antibody (T4).
  • tau protein or tau protein or tau protein from a sample collected from a subject by an antigen-antibody reaction using two types of antibodies that specifically bind to tau protein or phosphorylated tau protein is detected.
  • a tau protein detection method for detecting a phosphorylated tau protein wherein the sample is a blood sample and one of two types of antibodies is used fixed to a carrier or a molecule capable of binding to the carrier. The first antibody is labeled with, and the other is the second antibody, which is labeled without being immobilized on a carrier, and the tau protein is applied to the N-terminal region, the 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 sequence contained in the intermediate region or the amino acid sequence contained in the N-terminal region, and one of the two types of the antibodies can be used.
  • the configuration is such that the blood sample is first subjected to an antigen-antibody reaction, and then the other is subjected to an antigen-antibody reaction with the blood sample.
  • the tau protein or phosphorylated tau protein in the sample is obtained by the sandwich method in which the first antibody substantially immobilized and the second antibody labeled without immobilization are combined. Is detected.
  • the first antibody and the second antibody an antibody having an epitope in the C-terminal region of the tau protein is not used, but an antibody having an epitope in the intermediate region or the N-terminal region is used, and the first antibody is immobilized.
  • a non-immobilized carrier that does not immobilize the antibody Do not use together.
  • 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 widely and preferably in various application fields utilizing the detection of tau protein or phosphorylated tau protein. be able to.

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TOM VANDEBROEK ET AL.: "Identification and Isolation of a Hyperphosphorylated, Conformationally Changed Intermediate of Human Pro tein Tau Expressed in Yeast", BIOCHEMISTRY, vol. 44, 2005, pages 11466 - 11475

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EP4409299A4 (en) * 2021-10-01 2025-08-13 Integrated Neurologics LLC METHODS AND DEVICES FOR DETECTING CEREBROSPINAL FLUID LEAKAGE
JP7450995B1 (ja) 2023-04-13 2024-03-18 国立研究開発法人量子科学技術研究開発機構 被験者の脳のタウタンパク質の蓄積量を推定する方法及び装置
WO2024214588A1 (ja) * 2023-04-13 2024-10-17 国立研究開発法人量子科学技術研究開発機構 被験者の脳のタウタンパク質の蓄積量を推定する方法及び装置
JP2024152000A (ja) * 2023-04-13 2024-10-25 国立研究開発法人量子科学技術研究開発機構 被験者の脳のタウタンパク質の蓄積量を推定する方法及び装置

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