Method for detecting the presence or amount of pt205 tau in a biological sample

Abstract

To provide a method for detecting the presence or amount of pT205Tau in a biological sample.SOLUTION: What is claimed is: A method for detecting the presence or amount of pT205Tau in a biological sample comprising contacting the biological sample with an anti- pT205Tau monoclonal or pT205Tau binding fragment thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods for detecting the presence or amount of pT205 Tau in a biological sample. [Background technology]

[0002] It is becoming clear that tau phosphorylation causes the accumulation of tau and amyloid beta and is involved in the onset and severity of Alzheimer's disease. The phosphorylation sites of tau involved in Alzheimer's disease have mainly been investigated, namely, threonine 181 (T181) and threonine 217 (T217).

[0003] In recent years, threonine 205 (T205) has been attracting attention as a phosphorylation site different from those that have traditionally been of interest. It has been suggested that tau phosphorylated at T205 (pT205 Tau, p-tau205), unlike the previously noted phosphorylations at T181 and T217, may be related to the pathology of both tau accumulation and amyloid beta accumulation (Non-Patent Document 1). Furthermore, Non-Patent Document 2 describes a study using mass spectrometry in a large cohort to investigate the relationship between various biomarkers and the stage of Alzheimer's disease, and as a result, it is described that elevated levels of pT205 Tau in plasma were observed in relatively late stages of Alzheimer's disease.

[0004] Regarding the detection of phosphorylated tau, for example, Non-Patent Document 3 describes that an assay by Simoa Technology (Quanterix) using a polyclonal antibody against phosphorylated tau detected tau phosphorylated at T202 or T205 in human cerebrospinal fluid, and that the measurement results of pT205 Tau showed a positive correlation with the measurement results of amyloid beta and tau by PET, indicating that pT205 Tau in human cerebrospinal fluid is strongly correlated with the accumulation of amyloid beta and tau in the brain. Furthermore, since the use of measurement methods using human cerebrospinal fluid and PET methods is limited due to their high invasiveness and high cost, a highly reliable method for measuring pT205 Tau in plasma is desired to achieve greater usability and cost efficiency. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Clifford R Jack Jr et al.,"Revised criteria for diagnosis and staging of Alzheimer's disease:Alzheimer's Association Workgroup", Alzheimer's Dement. 2024;1-27. [Non-patent document 2] Barthelemy, NR, Saef, B.,Li, Y., et al., CSF tau phosphorylation occupancies at T217 and T205 represent improved biomarkers of amyloid and tau pathology in Alzheimer's disease. Natureaging 3, 391-401 (2023). [Non-patent document 3] Juan Lantero-Rodriguez et al., "CSF p-tau205: a biomarker of tau pathology in Alzheimer's disease", ActaNeuropathol. 2024;147(1):12. [Non-patent document 4] Barthelemy, NR, Horie, K.,Sato, C. & Bateman, RJ Blood plasma phosphorylated-tau isoforms trackCNS change in Alzheimer's disease. J Exp Med 217, doi:10.1084 / jem.20200861(2020). Summary of the Invention [Problem to be solved by the invention]

[0006] In the method described in Non-Patent Document 3, detection of tau phosphorylated at T205 using purified peptides was observed to cross-react with tau phosphorylated at T202. Furthermore, although the quantification results of pT205 Tau by the method described in Non-Patent Document 3 were positively correlated with the quantification results of pT205 Tau by mass spectrometry, the correlation between the individual values ​​was not necessarily linear. Thus, the method described in Non-Patent Document 3 had room for improvement in the accuracy of detecting the presence or amount of pT205 Tau.

[0007] The present disclosure aims to provide a method for detecting the presence or amount of pT205 Tau in a biological sample. Another object of the present disclosure is to provide a method for detecting the presence or amount of pT205 Tau in a biological sample with high accuracy. [Means for solving the problem]

[0008] The present inventors have found that the presence or amount of pT205 Tau in a biological sample can be detected with high accuracy by a method comprising the step of contacting the biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof.

[0009] The present disclosure relates, for example, to the following: [1] A method for detecting the presence or amount of pT205 Tau in a biological sample, comprising: contacting said biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof; A method comprising: [2] A method for assessing tau accumulation in a subject, comprising: contacting the subject's biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. A method comprising: [3] A method for assessing amyloid beta accumulation in a subject, comprising: contacting the subject's biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. A method comprising: [4] The method according to any one of [1] to [3], further comprising the step of contacting a complex formed between the anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment and the pT205 Tau with a labeled detection antibody that binds to the pT205 Tau. [5] The method according to [4], further comprising the step of detecting the presence or amount of a complex formed between the anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment, the pT205 Tau and the labeled detection antibody. [6] The method according to any one of [1] to [5], wherein the presence or amount of the complex is detected by ELISA. [7] The method according to [6], wherein the anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment is bound to beads, and the enzyme reaction of the reporter enzyme in the ELISA is carried out in a system containing one particle of the bead. [8] The method according to any one of [1] to [7], wherein the biological sample is obtained from a subject suffering from or at risk of suffering from Alzheimer's disease or mild cognitive impairment due to Alzheimer's disease. [9] The method according to any one of [1] to [8], wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

[10] The method according to any one of [1] to [9], wherein the biological sample is cerebrospinal fluid.

[11] The method according to any one of [1] to [9], wherein the biological sample is blood, serum, or plasma.

[12] The method according to any one of [5] to

[11] , further comprising a step of comparing the amount of the complex detected using the biological sample with the amount of the complex detected using a control biological sample.

[13] A method for diagnosing Alzheimer's disease, comprising detecting the presence or amount of pT205 Tau in a biological sample, assessing the accumulation of Tau in a subject, or assessing the accumulation of amyloid beta in a subject, according to the method described in any one of [1] to

[12] .

[14] A method for obtaining data for use in diagnosing Alzheimer's disease, comprising detecting the presence or amount of pT205 Tau in a biological sample, assessing the accumulation of Tau in a subject, or assessing the accumulation of amyloid beta in a subject, according to the method described in any one of [1] to

[12] .

[15] A method for assisting in the diagnosis of Alzheimer's disease, comprising detecting the presence or amount of pT205 Tau in a biological sample, assessing the accumulation of Tau in a subject, or assessing the accumulation of amyloid beta in a subject, according to the method described in any one of [1] to

[12] .

[16] A kit for detecting the presence or amount of pT205 Tau, comprising: Anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment Includes a kit.

[17] A kit for assessing tau accumulation, comprising: Anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment Includes a kit.

[18] A kit for assessing amyloid beta accumulation, comprising: Anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment Includes a kit.

[19] A kit according to any one of

[16] to

[18] for use in any one of the methods of [1] to

[15] .

[20] The kit according to any one of

[16] to

[19] , which comprises a reagent or instrument that can be used in detecting the presence or amount of pT205 Tau, or instructions for using the kit. [Effects of the Invention]

[0010] According to the present disclosure, there is provided a method capable of detecting the presence or amount of pT205 Tau in a biological sample. Also, according to the present disclosure, there is provided a method capable of detecting the presence or amount of pT205 Tau in a biological sample with high accuracy. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the absorbance when each antibody was used in Example 1. [Figure 2] FIG. 2 shows the results of performance evaluation of each antibody using Simoa when each commercially available p-tau205 antibody was bound to magnetic beads and combined with biotin-labeled Tau12 in Example 2. [Figure 3] FIG. 3 shows the results of measuring the amount of p-tau205 in CSF from a control and an AD patient by Simoa p-tau205 assay in Example 3. [Figure 4]FIG. 4 shows the correlation between cognitive function score (MMSE) and p-tau205 amount in CSF of AD patients in Example 3. [Figure 5] FIG. 5 shows the results of measuring the amount of p-tau205 in human plasma from a control and an AD patient by the Simoa p-tau205 assay in Example 4. [Figure 6] FIG. 6 shows the results of Example 5, comparing the amounts of p-tau205 measured by the Simoa p-tau205 assay and LC / MS in the CSF of a control and an AD patient. DETAILED DESCRIPTION OF THE INVENTION

[0012] Modes for carrying out the present disclosure will be described below, but the present disclosure should not be construed as being limited to the following embodiments.

[0013] In the present disclosure, the terms "p<single-letter amino acid notation: X><number: Y> Tau" and "p-tau<number: Y>" refer to tau in which the amino acid residue corresponding to the amino acid represented by X at position Y in the amino acid sequence of 2N4R tau shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_005901.2) is phosphorylated. For example, terms such as "pT205 Tau" and "p-tau205" refer to tau in which the amino acid residue corresponding to the threonine at position 205 in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated. Furthermore, for example, terms such as "pS202 / pT205 Tau" and "p-tau(S202,T205)" refer to tau in which the amino acid residues corresponding to the serine at position 202 and the threonine at position 205 in the amino acid sequence shown in SEQ ID NO: 1 are phosphorylated. In one aspect, "pXY Tau" and "p-tauY" may refer to tau in which at least the amino acid residue corresponding to the Yth amino acid represented by X in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated, and other amino acid residues may be further phosphorylated.

[0014] "pXY Tau" and "p-tauY" according to one embodiment are not limited to tau itself in which threonine at position 205 in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated. That is, while multiple isoforms of tau exist, "pXY Tau" and "p-tauY" according to one embodiment may be a tau isoform other than 2N4R tau in which an amino acid residue corresponding to the amino acid at position Y, represented by X, in the amino acid sequence shown in SEQ ID NO: 1, which is the sequence of 2N4R tau, is phosphorylated. Furthermore, "pXY Tau" and "p-tauY" according to another embodiment may be tau in which threonine at position 205 in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated.

[0015] In the present disclosure, the anti-pT205 Tau monoclonal antibody may be any monoclonal antibody that specifically binds to pT205 Tau, and may be a commercially available antibody or an obtained antibody.

[0016] A commercially available monoclonal antibody that specifically binds to pT205 Tau may be, for example, Phospho-Tau(Thr205)(E7D3E) Rabbit mAb (BSA and Azide Free) (Cell Signaling Technology, Cat: 20739), Phospho-Tau(Thr205)(E7D3E) Rabbit mAb (Cell Signaling Technology, Cat: 49561), or Anti-Tau(phospho T205) antibody [EPR23505-13] (abcam, Cat: ab275027; monoclonal antibody). From the viewpoint of easily achieving high detection sensitivity, the anti-pT205 Tau monoclonal antibody according to a further embodiment may be Phospho-Tau(Thr205)(E7D3E) Rabbit mAb (BSA and Azide Free) (Cell Signaling Technology, Cat: 20739).

[0017] Anti-pT205 Tau monoclonal antibodies may be obtained, for example, according to the following procedure. First, pT205 Tau is administered multiple times over several weeks to immunize experimental animals such as mice. After confirming by ELISA or other methods that antibodies recognizing pT205 Tau have been produced in the serum of the immunized animals, antibody-producing cells (B cells) are collected from the animals. The antibody-producing cells (B cells) are collected, for example, from the spleen, lymph nodes, bone marrow, or blood collected from the animals. The collected B cells are fused with myeloma cells according to a standard method to produce hybridoma cells. Antibodies produced by each of the produced hybridoma cells are evaluated using methods such as ELISA or Western blotting, and a clone producing an antibody that specifically binds to pT205 Tau is selected from the hybridoma cells. Anti-pT205 Tau monoclonal antibodies are obtained as antibodies produced by the hybridoma cells selected in this manner.

[0018] In the present disclosure, the pT205 Tau-binding fragment of an anti-pT205 Tau monoclonal antibody may be any fragment of the anti-pT205 Tau monoclonal antibody that specifically binds to pT205 Tau, and may be, for example, Fab, F(ab')2, scFv, dsFv, or VHH. These pT205 Tau-binding fragments may be obtained by standard methods. For example, Fab and F(ab')2 may be obtained by cleaving an anti-pT205 Tau monoclonal antibody with an enzyme (e.g., papain or pepsin). For example, scFv and dsFv may be isolated and purified after being artificially expressed in bacteria, cells, etc. For example, VHH may be obtained by cloning a VHH gene using mRNA from B cells collected from a camelid immunized with pT205 Tau, and then artificially expressing the gene in bacteria, cells, etc., and then isolating and purifying the VHH.

[0019] In the present disclosure, a biological sample is a sample obtained from a subject that contains or may contain tau. The subject is, for example, a mammalian subject, preferably a human subject. The biological sample may be a sample collected from a subject or a processed product thereof. For example, the biological sample may be blood, serum, plasma, or cerebrospinal fluid. In one embodiment, the biological sample may be cerebrospinal fluid, which is rich in tau and therefore makes it easy to detect pT205 Tau. In another embodiment, the biological sample may be blood, serum, or plasma, which allows the method of the present disclosure to detect pT205 Tau with high sensitivity, making it easy to detect the presence or amount of pT205 Tau even when using a blood sample, which can be collected less invasively from tissue other than neural tissue.

[0020] A biological sample according to one embodiment may be obtained from a subject suffering from or at risk of suffering from Alzheimer's disease or mild cognitive impairment due to Alzheimer's disease. The present inventors found that an assay according to one embodiment of the present disclosure results in a greater signal derived from pT205 Tau in a biological sample from an Alzheimer's disease patient than in a biological sample from a control subject. Therefore, when a biological sample according to one embodiment is obtained from a subject suffering from or at risk of suffering from Alzheimer's disease or mild cognitive impairment due to Alzheimer's disease, it is believed that an assay according to one embodiment can be used to diagnose, assist in the diagnosis of, or obtain data for use in the diagnosis of Alzheimer's disease. That is, one embodiment of the present disclosure may be a method for diagnosing Alzheimer's disease, a method for assisting in the diagnosis of Alzheimer's disease, or a method for obtaining data for use in the diagnosis of Alzheimer's disease. These methods can obtain data for diagnosing, assisting in the diagnosis of, or using for the diagnosis of the severity of Alzheimer's disease in a subject, in addition to determining whether or not the subject has Alzheimer's disease. Furthermore, one embodiment of the present disclosure may be a method for treating Alzheimer's disease or mild cognitive impairment caused by Alzheimer's disease, comprising detecting the presence or amount of pT205 Tau in a biological sample, and administering a therapeutic agent for Alzheimer's disease to a subject from whom the biological sample was derived that was found to contain the presence or a predetermined amount of pT205 Tau.

[0021] Next, a method for detecting the presence or amount of pT205 Tau in a biological sample according to a first embodiment of the present disclosure (hereinafter also referred to as the "detection method of the first embodiment") will be described. The detection method according to one aspect of the first embodiment includes, in this order, a step of contacting a biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof (contacting step), a step of contacting a labeled detection antibody that binds to pT205 Tau with a complex formed between the anti-pT205 Tau monoclonal antibody or the pT205 Tau binding fragment and pT205 Tau (labeling step), and a step of detecting the presence or amount of a complex formed between the anti-pT205 Tau monoclonal antibody or the pT205 Tau binding fragment, pT205 Tau and the labeled detection antibody.

[0022] In the contacting step, the biological sample is contacted with an anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof. This allows the pT205 Tau in the biological sample and the anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof to form a complex. The contacting step is performed by allowing the biological sample and the anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof to coexist in the same solution. The specific contacting conditions may be any conditions that allow the formation of the complex. For example, the concentrations of the biological sample and the anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof, the contact temperature, the contact time, the solvent, and the solutes not directly involved in the formation of the complex can be appropriately selected and optimized by those skilled in the art within the ranges that allow the formation of the complex. Furthermore, the anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment used in the contact step may be labeled with, for example, another substance, or may be labeled with a reporter enzyme or reporter dye, etc., depending on the measurement method in the detection step described below.

[0023] The contacting step forms a complex between the anti-pT205 Tau monoclonal antibody or pT205 Tau-binding fragment and pT205 Tau (hereinafter also referred to as "pT205 Tau-antibody (fragment) complex"). Such a complex can be measured according to a method used to detect an antigen using an antibody or its antigen-binding fragment, for example, ELISA (enzyme-linked immunosorbent assay), Western blotting, or immunofluorescence, or can also be measured by these methods combined with immunoprecipitation or lateral flow assay. These measurement methods can be appropriately performed by those skilled in the art based on their common general technical knowledge. From the viewpoint of excellent sensitivity, the pT205 Tau-antibody (fragment) complex may be measured by ELISA. In this embodiment, the case where the measurement method is ELISA (also known as sandwich ELISA) will be described in detail.

[0024] When the measurement method is ELISA, the anti-pT205 Tau monoclonal antibody or the pT205 Tau binding fragment is bound to a carrier. The binding of the anti-pT205 Tau monoclonal antibody or the pT205 Tau binding fragment to the carrier may be carried out, for example, by incubating the carrier in contact with a solution containing the antibody or the pT205 Tau binding fragment, and the carrier may then be washed as necessary. The carrier in ELISA may be any carrier that a person skilled in the art would use, for example, in the form of particles or plates, and the particle-shaped carrier may be, for example, beads.

[0025] In the labeling step, a complex formed between an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment and pT205 Tau (pT205 Tau-antibody (fragment) complex) is contacted with a labeled detection antibody that binds to pT205 Tau. As a result, in the labeling step, a complex in which the pT205 Tau-antibody (fragment) complex is further labeled with the labeled detection antibody (hereinafter also referred to as a "labeled pT205 Tau-antibody (fragment) complex") is formed.

[0026] The labeled detection antibody is an antibody that directly or indirectly detectably labels the labeled pT205 Tau-antibody (fragment) complex with a signal. The labeled detection antibody may be (i) a conjugated protein of an antibody that recognizes an amino acid other than T205 of tau and does not interfere with the anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment in binding to pT205 Tau, and a reporter enzyme, or (ii) an antibody that recognizes an amino acid other than T205 of tau and does not interfere with the anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment in binding to pT205 Tau, labeled with an affinity tag (e.g., biotin). When the labeled detection antibody is the conjugated protein (i) above, the labeling step may be carried out by contacting the pT205 Tau-antibody (fragment) complex on the surface of the carrier under the same conditions as in one embodiment of the contacting step. For example, when the labeled detection antibody is the labeled antibody described in (ii) above, the labeling step may be carried out by first contacting the labeled detection antibody with the pT205 Tau-antibody (fragment) complex on the surface of the carrier. In this case, after the labeling step and before the detection step, the labeled pT205 Tau-antibody (fragment) complex is further contacted with a conjugated protein consisting of the affinity tag, a high-affinity protein (e.g., streptavidin), and a reporter enzyme. This allows the reporter enzyme to be modified onto the labeled detection antibody in the labeled pT205 Tau-antibody (fragment) complex.

[0027] The reporter enzyme may be one commonly used as a reporter enzyme in ELISA, such as β-galactosidase or luciferase. The substrate for the reporter enzyme may be any that emits light or increases fluorescence intensity upon enzymatic reaction of the reporter enzyme, and can be appropriately selected by those skilled in the art depending on the type of reporter enzyme.

[0028] In the detection step, the presence or amount of a complex (labeled pT205 Tau-antibody (fragment) complex) formed between the anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment, pT205 Tau, and the labeled detection antibody is detected. That is, in the detection step, the labeled pT205 Tau-antibody (fragment) complex is measured to obtain information on its presence or amount. Thereby, the presence or amount of pT205 Tau in a biological sample can be detected using the amount of the labeled pT205 Tau-antibody (fragment) complex as an indicator. In the detection step, a substrate for a reporter enzyme that the labeled detection antibody has or that modifies the labeled detection antibody is added to the labeled pT205 Tau-antibody (fragment) complex, and the labeled pT205 Tau-antibody (fragment) complex can be measured based on the signal generated by the enzymatic reaction of the reporter enzyme.

[0029] The conditions for adding a substrate for a reporter enzyme and measuring a signal generated by the enzymatic reaction of the reporter enzyme may be any conditions that allow significant detection of the generated signal. For example, the concentration, measurement temperature, measurement time, solvent, solute not directly involved in the enzymatic reaction, and measurement equipment can be appropriately selected and optimized by those skilled in the art.

[0030] In one embodiment where the carrier is a bead, the anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof may be bound to the bead, and the enzymatic reaction of the reporter enzyme in ELISA may be carried out in a system containing one bead particle. In this case, the labeled pT205 Tau-antibody (fragment) complex is concentrated in the system containing one bead particle, which tends to increase the detection sensitivity of pT205 Tau. In this case, the volume of the system containing one bead particle may be, for example, 1 μL or less, 1 nL or less, 100 pL or less, 10 pL or less, 1 pL or less, or 100 fL or less, and in a further embodiment, 100 fL or less. Such a system containing a single bead can be realized, for example, by loading a bead suspension into each well of a well plate whose number and volume are set so that less than one bead is stochastically arranged, by pouring an aqueous solution containing suspended beads into each well, and then sealing each well with a water-immiscible organic solvent. A technology that can realize the volume of a system according to one embodiment is known, such as Quanterix's Simoa® Bead Assay Technology (Simoa HD-X / SR-X). In a further embodiment, the presence or amount of the labeled pT205 Tau-antibody (fragment) complex in the detection step may be detected using the Simoa Bead Assay Technology. In a further embodiment, the presence or amount of the labeled pT205 Tau-antibody (fragment) complex in the detection step may be detected by a digital ELISA method. In the digital ELISA method, the amount of the target substance is evaluated using the number of systems that produce a detectable signal as an index, based on the fact that a detectable signal is generated by the enzymatic reaction of the reporter enzyme only in systems in which the target substance is present, among the systems containing a single bead as described above.

[0031] In the detection method of the first embodiment, in the contacting step, the biological sample is contacted with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof, thereby enabling detection of pT205 Tau in the biological sample with higher accuracy than when a polyclonal antibody is used.

[0032] A second embodiment of the present disclosure relates to a method for assessing tau accumulation in a subject (hereinafter also referred to as the "tau accumulation assessment method of the second embodiment"). "Assessing tau accumulation in a subject" refers to assessing the amount of tau accumulation in the brain of the subject or assessing whether the subject is tau positive or negative. In one aspect, the tau accumulation assessment method of the second embodiment may include, in this order, a contacting step, a detecting step, and a step of comparing the amount of complex detected using a biological sample with the amount of complex detected using a control biological sample (comparison step). In this case, the contacting step and the detecting step can be performed in the same manner as the contacting step and the detecting step in the detection method of the first embodiment. It has been reported that phosphorylation of T205 of tau is positively correlated with tau accumulation (Non-Patent Document 3). Therefore, it is believed that tau accumulation can be assessed by measuring pT205 Tau.

[0033] In the comparison step, the amount of labeled pT205 Tau-antibody (fragment) complex detected using the biological sample to be evaluated (hereinafter also referred to as the "biological sample of interest") is compared with the amount of labeled pT205 Tau-antibody (fragment) complex detected using a control biological sample. The control biological sample may be of the same type as the biological sample of interest (e.g., blood, serum, plasma, or cerebrospinal fluid) and known to have no tau accumulation (negative control) and / or known to have tau accumulation (positive control). In one embodiment, the control may be a negative control of the same type as the biological sample of interest. The negative control may be, for example, a subject with healthy cognitive function or a subject not suffering from Alzheimer's disease. The positive control may be, for example, a subject with Alzheimer's disease or a subject with stage 4 or later of Alzheimer's disease. Whether the subject corresponds to a negative control or a positive control may be determined, for example, by a cognitive function score (MMSE). Furthermore, the amount of labeled pT205 Tau-antibody (fragment) complex detected using a control biological sample may be a value measured by the same method at the same time as detecting the amount of labeled pT205 Tau-antibody (fragment) complex using the biological sample of interest, or may be a reference value measured at a different time from detecting the amount of labeled pT205 Tau-antibody (fragment) complex using the biological sample of interest. Furthermore, in the comparison step, instead of the amount of labeled pT205 Tau-antibody (fragment) complex itself, a measurement value or parameter that is proportional to the amount of labeled pT205 Tau-antibody (fragment) complex may be compared.

[0034] The comparison step may indicate, for example, that tau has accumulated or is likely to have accumulated in a subject from which a biological sample of interest satisfying (A1), (A2), or (A3) below was derived, and in a further aspect, may indicate that tau has accumulated or is likely to have accumulated in a subject from which a biological sample of interest satisfying (A2) or (A3) below was derived. (A1) The amount of labeled pT205 Tau-antibody (fragment) complex detected using the biological sample of interest is closer to the average amount of labeled pT205 Tau-antibody (fragment) complex detected using the positive control biological sample than to the average amount of labeled pT205 Tau-antibody (fragment) complex detected using the negative control biological sample. (A2) The amount of labeled pT205 Tau-antibody (fragment) complex detected using the biological sample of interest is greater than the upper limit of a certain percentage confidence interval (e.g., 68% confidence interval or 95% confidence interval) when the amount of labeled pT205 Tau-antibody (fragment) complex detected using the negative control biological sample is fitted according to a normal distribution. (A3) The amount of labeled pT205 Tau-antibody (fragment) complex detected using the biological sample of interest is greater than the lower limit of a specified percentage confidence interval (e.g., 68% confidence interval or 95% confidence interval) when the amount of labeled pT205 Tau-antibody (fragment) complex detected using the positive control biological sample is fitted according to a normal distribution.

[0035] A third embodiment of the present disclosure relates to a method for assessing amyloid beta accumulation in a subject (hereinafter also referred to as the "third embodiment amyloid beta accumulation assessment method"). "Assessing amyloid beta accumulation in a subject" refers to assessing the amount of amyloid beta accumulation in the subject's brain or assessing whether the subject is amyloid beta positive or negative. In one aspect, the amyloid beta accumulation assessment method of the third embodiment may include a contacting step, a detecting step, and a comparing step, in this order. In this case, the contacting step, the detecting step, and the comparing step can be performed in the same manner as the contacting step, the detecting step, and the comparing step in the tau accumulation assessment method of the second embodiment. It has been reported that phosphorylation of T205 of tau is positively correlated with amyloid beta accumulation (Non-Patent Document 3). Therefore, it is believed that amyloid beta accumulation can be assessed by measuring pT205 Tau. For example, the comparison step may indicate that amyloid beta has accumulated or is likely to have accumulated in a subject from which a biological sample of interest satisfying (A1), (A2) or (A3) above was derived, and in a further aspect, may indicate that amyloid beta has accumulated or is likely to have accumulated in a subject from which a biological sample of interest satisfying (A2) or (A3) above was derived.

[0036] A fourth embodiment of the present disclosure is a kit for detecting the presence or amount of pT205 Tau, the kit comprising an anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof. The kit of the fourth embodiment can be used to detect the presence or amount of pT205 Tau according to one aspect of the detection method of the first embodiment. A kit according to one aspect of the fourth embodiment may be for use in the detection method of the first embodiment. The kit according to one aspect of the fourth embodiment may further comprise a reagent or instrument usable in detecting the presence or amount of pT205 Tau, or instructions for using the kit. Another aspect of the fourth embodiment may be use of an anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof, or a kit comprising the same, in the detection method of the first embodiment.

[0037] A fifth embodiment of the present disclosure is a kit for assessing tau accumulation, comprising an anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof. The kit of the fifth embodiment can be used to assess tau accumulation in accordance with one aspect of the method for assessing tau accumulation of the second embodiment. A kit according to one aspect of the fifth embodiment may be for use in the method for assessing tau accumulation of the second embodiment. The kit according to one aspect of the fifth embodiment may further comprise a reagent or instrument that can be used in detecting the presence or amount of pT205 Tau, or instructions for using the kit. Another aspect of the fifth embodiment may be use of an anti-pT205 Tau monoclonal antibody or a pT205 Tau-binding fragment thereof, or a kit comprising the same, in the method for assessing tau accumulation of the second embodiment.

[0038] A sixth embodiment of the present disclosure is a kit for assessing amyloid beta accumulation, the kit comprising an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. The kit of the sixth embodiment can be used to assess amyloid beta accumulation according to one aspect of the method for assessing amyloid beta accumulation of the third embodiment. A kit according to one aspect of the sixth embodiment may be for the method for assessing amyloid beta accumulation of the third embodiment. The kit according to one aspect of the sixth embodiment may further comprise a reagent or instrument that can be used in detecting the presence or amount of pT205 Tau, or instructions for using the kit. Another aspect of the sixth embodiment may be use of an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof, or a kit comprising the same, in the method for assessing amyloid beta accumulation of the third embodiment. [Example]

[0039] The present disclosure will be described in more detail below using examples, but the present disclosure should not be construed as being limited to the following examples.

[0040] Example 1: Evaluation of reactivity of commercially available anti-p-tau205 antibodies Six peptides, shown in Table 1, were synthesized by combining phosphorylation of amino acid residues S202, T205, and S208 in the amino acid sequence corresponding to residues 200 (P) to 211 (R) of human 2N4R tau. The N-terminus of each peptide was biotinylated. Three commercially available anti-p-tau205 antibodies were used: Phospho-Tau (Thr205) (E7D3E) Rabbit mAb (Cell Signaling Technology, Cat: 20739SF), Anti-Tau (phospho T205) antibody [EPR23505-13] (Abcam, Cat: ab275027; monoclonal antibody), and Phospho-Tau (Thr205) Polyclonal Antibody (Thermo Fisher Scientific, Cat: PA5-121265). As a control, we also used the anti-pS202 / pT205 antibody (specific for tau phosphorylated at both S202 and T205), Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8) (Thermo Fisher Scientific, Cat: MN1020).

[0041] [Table 1]

[0042] Each peptide was dissolved in PBS + 0.05% Tween 20 (PBS supplemented with 0.05% Tween 20) and diluted with ELISA reaction solution (PBS, 0.5% BSA, 0.05% Tween 20) to prepare a 2 μg / mL solution. 100 μL / well of the 2 μg / mL peptide solution was applied to a streptavidin-coated plate (Thermo Fisher Scientific, Cat: 15121). After incubating at 25°C for 30 minutes, the plate was washed three times with PBS + 0.05% Tween 20. 100 μL / well of each antibody solution, diluted with ELISA reaction solution to 100 ng / mL, was applied and incubated at room temperature for 1 hour. The plate was then washed three times with PBS + 0.05% Tween 20. Next, 100 μL / well of 5000-fold diluted Peroxidase-conjugated Goat anti-Mouse IgG (Jackson Immuno Research, Cat. No. 115-035-166) was applied to the AT8-applied wells, and 100 μL / well of 5000-fold diluted Peroxidase-conjugated Goat anti-Rabbit IgG (Jackson Immuno Research, Cat. No. 111-035-144) was applied to the other wells. After washing three times with PBS + 0.05% Tween 20, 100 μL / well of TMB substrate solution (prepared by mixing TMB Peroxidase Substrate (KPL, Cat. No. 5120-0047) and Peroxidase Substrate Solution B (KPL, Cat. No. 5120-0037) at a 1:1 (v / v) ratio) was added to initiate the color reaction. After 10 minutes, the reaction was stopped by adding 100 μL / well of 0.5 M sulfuric acid, and the absorbance at 450 nm (secondary wavelength 650 nm) was read using a microplate reader SpectraMax190 (Molecular Devices).

[0043] Figure 1 shows the absorbance measured using each antibody. The polyclonal antibody from Thermofisher also showed reactivity to phosphorylated S202 and S208, whereas the monoclonal antibodies from CST (Cell Signaling Technology) and Abcam showed high specificity for phosphorylated T205. These results suggest that the assay using monoclonal antibodies exhibits higher specificity for pT205 tau than the known assay using polyclonal antibodies. The control AT8 reacted only with peptides phosphorylated at both S202 and T205, a result consistent with literature reports. Therefore, the evaluation system was deemed valid.

[0044] Example 2: Comparison of reactivity of commercially available p-tau205 antibodies with Simoa Using the Simoa Homebrew Assay Development Kit (Quanterix, Cat. No. 101354), Phospho-Tau (Thr205) (E7D3E) Rabbit mAb (Cell Signaling Technology, Cat. No. 20739SF), Anti-Tau (phospho T205) antibody [EPR23505-13] (Abcam, Cat. No. ab275027; monoclonal antibody), and Phospho-Tau (Thr205) Polyclonal Antibody (Thermo Fisher Scientific, Cat. No. PA5-121265) were conjugated to magnetic beads. The same kit was also used to biotinylate Tau12 (Biolegend, Cat. No. 806501). Antibody-conjugated beads and biotinylated antibodies were prepared according to the Quanterix manual.

[0045] Each antibody-conjugated bead was diluted with Beads Diluent (Quanterix, Cat. No. 101362) to prepare an antibody bead solution. Biotin-labeled Tau12 was diluted with Tau 2.0 Sample Diluent to 0.3 μg / mL to prepare a labeled detection antibody solution. Tau 441 DYRK1A phosphorylated antibody (SignalChem, Cat. No. T08-50RN) was diluted three-fold starting from 200 pg / mL with Tau 2.0 Sample Diluent (Quanterix, Cat. No. 101556) (7 concentration points from 200 pg / mL to 0.82 pg / mL). The dilution series and a blank (Tau 2.0 Sample Diluent) were measured using a Simoa SR-X (Quanterix) with the antibody combinations listed in Table 2 below. The reactivity of commercially available p-tau205 antibodies with Simoa was compared by comparing the obtained signals (AEB: average number of enzymes per bead, a signal unit specific to Simoa). The measurement conditions are shown in Table 3 below. Incubation for the measurement was performed using a Simoa SR-X shaker (Quanterix). Washing steps between steps were performed using a BioTek-405 washer (Agilent) with the Simoa SR-X washing protocol installed.

[0046] [Table 2]

[0047] [Table 3]

[0048] Figure 2 shows the results of Simoa performance evaluation of commercially available p-tau205 antibodies conjugated to magnetic beads in combination with biotin-labeled Tau12. Compared with the Abcam monoclonal antibody and Thermofisher polyclonal antibody, the Cell Signaling Technology monoclonal antibody demonstrated superior reactivity. Based on these results, the "Simoa p-tau205 assay" was developed as a highly sensitive Simoa method for measuring p-tau205, combining magnetic bead-bound Phospho-Tau (Thr205) (E7D3E) Rabbit mAb (Cell Signaling Technology, Cat: 20739SF) with biotin-labeled Tau12 (Biolegend, Cat: 806501).

[0049] Example 3: Measurement of p-tau205 levels in CSF using the Simoa p-tau205 assay Cerebrospinal fluid (CSF) samples collected from controls (those with normal cognitive function) and AD patients (those with Alzheimer's disease) were purchased from PrecisionMed and measured using the Simoa p-tau205 assay developed in Example 2. Each CSF sample was diluted 5-fold with Tau 2.0 Sample Diluent, and 10 control and 15 AD patients were measured. Antibody-bound beads and biotin-labeled antibodies were prepared, and measurements using the Simoa SR-X were performed according to the method described in Example 2. A calibration curve was created from a dilution series of Tau 441 DYRK1A phosphorylated (SignalChem, Cat: T08-50RN), and the measured values ​​for each CSF sample were calculated. Significant differences between groups were tested using the Mann-Whitney test.

[0050] Figure 3 shows the results of measuring p-tau205 levels in CSF from control and AD patients using the Simoa p-tau205 assay. Figure 4 shows the correlation between cognitive function scores (MMSE) and p-tau205 levels in CSF from AD patients. As shown in Figure 3, when commercially available CSF samples were measured using the Simoa p-tau205 assay, p-tau205 levels in the AD group were significantly higher (p<0.05). Furthermore, when the correlation between cognitive function scores (MMSE) and p-tau205 was analyzed for AD patient samples, a significant negative correlation was observed (Spearman's rho=-0.524, p<0.05), as shown in Figure 4. These results are consistent with the fact that CSF p-tau205 is thought to reflect tau accumulation in the brain and that tau accumulation is well known to correlate with cognitive decline.

[0051] Example 4: Measurement of p-tau205 levels in plasma using the Simoa p-tau205 assay Human plasma samples from control (cognitively normal) and AD patients (Alzheimer's disease patients) were purchased from PrecisionMed, and p-tau205 measurements were performed using the Simoa p-tau205 assay. Each plasma sample was diluted 2-fold with Tau 2.0 Sample Diluent. Measurements were performed using magnetic bead-bound Phospho-Tau (Thr205) (E7D3E) Rabbit mAb (Cell Signaling Technology, Cat: 20739SF) and biotin-labeled Tau12 (Biolegend, Cat: 806501) with the Simoa SR-X according to the method described in Example 2.

[0052] Figure 5 shows the results of measuring the amount of p-tau205 in human plasma from control and AD patients using the Simoa p-tau205 assay. Although there have been no previous reports on measuring p-tau205 in human plasma using immunoassays, Figure 5 shows that the Simoa p-tau205 assay developed here was able to detect a significant signal and confirmed a tendency for the amount of p-tau205 to be higher in the AD group than in the control group.

[0053] Example 5: Evaluation of correlation between CSF measurement results of LC / MS p-tau205 assay and Simoa p-tau205 assay For the CSF samples (10 control and 15 AD cases) measured in Example 3, p-tau205 was measured by LC / MS with reference to Non-Patent Document 4. Specifically, anti-tau-1 antibody, clone PC1C6 (Sigma-Aldrich, Cat: MAB3420) and anti-tau antibody, clone Tau 12 (Sigma-Aldrich, Cat: MAB2241) were first bound to magnetic beads and immunoprecipitated from the CSF. Subsequently, the samples were eluted with perchloric acid, desalted, enzymatically digested, and purified, and the amount of p-tau205 was measured by LC / MS. The measurement results were compared with the amount of p-tau205 measured by the Simoa p-tau205 assay in Example 3.

[0054] Figure 6 shows the results of comparing the amount of p-tau205 measured by the Simoa p-tau205 assay and LC / MS in the CSF of control and AD patients. The LC / MS method can specifically detect p-tau205 by measuring its molecular weight. Figure 6 shows that the results of p-tau205 measurement in human cerebrospinal fluid using the Simoa p-tau205 assay were highly correlated with those measured by the LC / MS method (rho = 0.9554), confirming the high specificity and validity of the Simoa p-tau205 assay. Furthermore, the LC / MS method used in this study is similar to that used in Non-Patent Document 2, which evaluated the usefulness of p-tau205 in a large cohort. Therefore, the excellent correlation between LC / MS and Simoa p-tau205 demonstrated that the Simoa p-tau205 assay developed in this study is sufficiently robust for clinical use.

Claims

1. 1. A method for detecting the presence or amount of pT205 Tau in a biological sample, comprising: contacting the biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. A method comprising:

2. 2. The method of claim 1, further comprising contacting the complex formed between said anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment and said pT205 Tau with a labeled detection antibody that binds to said pT205 Tau.

3. 3. The method of claim 2, further comprising detecting the presence or amount of a complex formed between said anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment, said pT205 Tau and said labeled detection antibody.

4. The method of claim 3, wherein the detection of the presence or amount of the complex is performed by ELISA.

5. The method according to claim 4, wherein the anti-pT205 Tau monoclonal antibody or its pT205 Tau binding fragment is bound to a bead, and the enzyme reaction of the reporter enzyme in the ELISA is carried out in a system containing one particle of the bead.

6. The method according to any one of claims 1 to 5, wherein the biological sample is obtained from a subject suffering from or at risk of suffering from Alzheimer's disease or mild cognitive impairment due to Alzheimer's disease.

7. 1. A method for assessing tau accumulation in a subject, comprising: contacting the subject's biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. A method comprising:

8. 1. A method for assessing amyloid beta accumulation in a subject, comprising: contacting the subject's biological sample with an anti-pT205 Tau monoclonal antibody or a pT205 Tau binding fragment thereof. A method comprising:

9. The method according to any one of claims 1 to 8, wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

10. 1. A kit for detecting the presence or amount of pT205 Tau, comprising: Anti-pT205 Tau monoclonal antibody or pT205 Tau binding fragment thereof Includes a kit.

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