Method for estimating neurodegenerative state of brain

Abstract

Provided is a method capable of simply and accurately estimating a neurodegenerative state. This method for estimating the neurodegenerative state of the brain comprises: a measurement step for measuring a blood sample collected from a subject to obtain a plurality of measurement values for neurogranular protein-related peptides; and a use step in which a specific ratio value is calculated using the measured values of the plurality of neurogranular protein-related peptides, and the ratio value is used to determine the state of neurodegeneration.

Description

Technical Field

[0001] This invention relates to a method for inferring the neurodegenerative state of the brain. Background Technology

[0002] The number of people with dementia was estimated at approximately 50 million worldwide in 2017 and is projected to reach approximately 82 million by 2030. In Japan, the number is also projected to reach 7 million by 2025. Dementia is classified into neurodegenerative disorders, vascular dementia, and other causes. Neurodegenerative disorders account for the largest proportion, with Alzheimer's disease (AD), a well-known neurodegenerative disorder, accounting for more than half of all dementia cases. In the pathogenesis of Alzheimer's disease, amyloid-β (Aβ) peptide, produced by the cleavage of amyloid precursor proteins, has been reported to be closely related to the death of brain nerve cells. Therefore, Aβ peptide can be used as a biomarker for diagnosing the onset of Alzheimer's disease (Non-Patent Literature 1-2, Patent Literature 1-3).

[0003] Furthermore, neurogranin (Ng) is a protein abundantly distributed in the dendritic spines of brain nerve cells. Ng is a nerve-specific postsynaptic protein composed of 78 amino acid residues, which regulates calmodulin-dependent signaling and is associated with synaptic plasticity. Moreover, in Non-Patent Literature 3 and 4, Ng is also a biomarker of neurodegeneration and synaptic dysfunction, with increased concentrations reported in the cerebrospinal fluid (CSF) of AD patients. Non-Patent Literature 5 reports that Ng fragmentation is promoted in the brains of AD patients, and that Ng with post-translational modifications such as acetylation or glutathioneization is present. Furthermore, it is reported that the ratio of the total value of the three post-translational modified Ng1-78 to the Ng fragment peptide in CSF is increased in AD patients compared to the standard sample. Non-Patent Literature 6 and 7 report an increase in Ng48-76 in CSF compared to the standard sample in AD patients. In this way, Ng has been reported as a marker of neurodegeneration in AD patients in CSF.

[0004] Existing technical documents

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 6424757

[0007] Patent Document 2: Japanese Patent No. 6410810

[0008] Patent Document 3: Japanese Patent No. 6467512

[0009] Non-patent literature

[0010] Non-patent literature 1: Kaneko N et al.: Novel plasma biomarker surrogating cerebralamyloid deposition. (Proceedings of the National Academy of Sciences of Japan, Series B, Phys Biol Sci.) 2014; 90(9): 353-364.

[0011] Non-patent literature 2: Nakamura A et al.: High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018; 554(7691): 249-254.

[0012] Non-patent literature 3: Portelius E et al.: Cerebrospinal fluid neurogranin concentration in neurodegeneration: relation to clinical phenotypes and neuropathology. Acta Neuropathol. 2018; 136(3): 363-376.

[0013] Non-patent literature 4: Thorsell A et al.: Neurogranin in cerebrospinal fluid as a marker of synaptic degeneration in Alzheimer's disease. Brain Res. 2010; 1362: 13-22.

[0014] Non-patent literature 5: Kvartsberg H et al.: The intact postsynaptic protein neurogranin is reduced in brain tissue from patients with familial and sporadic Alzheimer's disease. Acta Neuropathol. 2019; 137(1): 89-102.

[0015] Non-patent literature 6: Kvartsberg H et al.: Characterization of the postsynaptic protein neurogranin in paired cerebrospinal fluid and plasma samples from Alzheimer's disease patients and healthy controls. Alzheimers Res Ther. 2015 Jul 1; 7(1): 40.

[0016] Non-patent literature 7: Kvartsberg H et al.: Cerebrospinal fluid levels of the synaptic protein neurogranin correlates with cognitive decline in prodromal Alzheimer's disease. (Alzheimer's Dement. 2015; 11(10): 1180-90) Summary of the Invention

[0017] The technical problem that the invention aims to solve

[0018] However, methods using biomarkers from the CSF to diagnose AD patients are highly invasive due to the need for CSF removal. Therefore, there is a demand for analyses based on less invasive and readily obtainable blood tests and their biomarkers. However, little has been analyzed for Ng and its fragment peptides in the blood, and their effectiveness as biomarkers for neurodegenerative dementia has not been reported.

[0019] Furthermore, the inventors' research on Ng and its fragment peptides in blood samples revealed that specific Ng and its fragment peptides may increase or decrease in patients with dementia caused by neurodegeneration, thus serving as useful biomarkers for the pathogenesis of this dementia. Further research indicated that blood samples were either frozen or stored at room temperature before analysis, and particularly after room temperature storage, Ng and its fragment peptides decomposed, causing changes in their quantity. Therefore, it was found that measuring only the increase or decrease of specific Ng or its fragment peptides does not account for the influence of room temperature storage, resulting in inaccurate measurement of the increase or decrease of Ng or its fragment peptides caused by dementia due to neurodegeneration, thus lacking accuracy in determining the state of neurodegeneration.

[0020] The purpose of this invention is to provide a simple and accurate method for predicting neurodegenerative states.

[0021] Solution to the above technical problems

[0022] The first aspect of the present invention relates to a method for inferring the neurodegenerative state of the brain, comprising: a measurement step, measuring a blood sample collected from a subject to obtain measurement values ​​of a plurality of neurogranule protein-related peptides; and a use step, using the value of at least one ratio of (1) to (11) described below from the plurality of neurogranule protein-related peptide measurement values ​​to determine the neurodegenerative state.

[0023] Invention Effects

[0024] According to the inference method of the first aspect of the present invention, the neurodegenerative state of a patient's brain can be easily and accurately inferred, and therefore can be used to determine whether there is a development of dementia caused by neurodegeneration. Attached Figure Description

[0025] Figure 1 A graph showing the rate of change of NI (Normalized Intensity) for subjects thawed and refrozen after approximately 3 hours at room temperature (subjects under RT3h-FT1 conditions) compared to the normalized intensity of subjects that did not undergo this process (subjects under control conditions).

[0026] Figure 2 Shown inFigure 1 The chart shows the distribution of the rate of change of all Ng ratios.

[0027] Figure 3 A graph showing the correlation between high Ng ratios ((1)–(4)) in dementia and FDG-PET scores is presented. The vertical axis represents the Ng ratio of each Ng peptide, and the horizontal axis represents the FDG-PET score.

[0028] Figure 4 The graph shows the correlation between the Ng ratio ((5) to (8)) indicating high levels of dementia and the FDG-PET score.

[0029] Figure 5 The graph shows the correlation between the Ng ratio ((9) to (11)) indicating high levels of dementia and the FDG-PET score.

[0030] Figure 6 A graph showing the correlation between the high Ng ratios ((1) to (4)) in dementia and VSRAD is presented. The vertical axis represents the Ng peptides, and the horizontal axis represents VSRAD.

[0031] Figure 7 The graph shows the correlation between the high Ng ratio ((5) to (8)) in dementia and VSRAD.

[0032] Figure 8 The graph shows the correlation between the high Ng ratio ((9) to (11)) in dementia and VSRAD.

[0033] Figure 9 A graph showing the correlation between the high Ng ratios ((1) to (4)) in dementia and MMSE is presented. The vertical axis represents the Ng ratio of each Ng peptide, and the horizontal axis represents MMSE.

[0034] Figure 10 The graph shows the correlation between the high Ng ratio ((5) to (8)) in dementia and MMSE.

[0035] Figure 11 The graph shows the correlation between the high Ng ratio ((9)~(11)) in dementia and MMSE. Detailed Implementation

[0036] 1. First Implementation

[0037] The proposed method of the first embodiment is a method for determining the neurodegenerative state of the brain, which includes a measurement step and a usage step.

[0038] 1-1. Measurement Procedure

[0039] In the measurement process, a blood sample is measured, and neurogranule protein-related peptides in the blood sample are analyzed. As an example of a preferred measurement process in the first embodiment, the blood sample is sequentially subjected to purification and mass spectrometry. That is, after purifying the blood sample containing blood collected from the subject, mass spectrometry is performed to obtain the measured value of neurogranule protein-related peptides in the blood sample. This method will be described in detail below.

[0040] (Purification process)

[0041] As a purification process for blood samples, affinity purification is a preferred example. This allows for the removal of impurities from the blood sample and the reliable detection of trace amounts of neuropeptides related to neurogranules in the blood.

[0042] In the first embodiment, the "neuronal granule protein-related peptide" (hereinafter referred to as "Ng peptide") includes, in addition to neurogranule proteins (Ng1-78) (serial number 36) and their fragment peptides, neurogranule proteins or their fragment peptides that have been modified by acetylation or cysteine ​​substitution. As an example of Ng peptide, the peptides described in Tables 3 and 4 in the embodiments described later can be cited.

[0043] As affinity purification, it can be a single purification or multiple purifications, but from the viewpoint of being able to detect Ng peptides with higher sensitivity, it is preferable to perform two purifications. In this case, the purification steps sequentially include a first binding step, a first washing step, a first elution step, a neutralization step, a second binding step, a second washing step, and a second elution step.

[0044] In the first binding step, the blood sample is brought into contact with the first carrier. Thereby, the Ng peptide in the blood sample binds to the first carrier, yielding the first conjugate.

[0045] A blood sample is a sample solution containing blood collected from a subject, preferably including blood itself or its dilutions. Blood samples include whole blood, plasma, and serum. A blood sample can be prepared by centrifuging, freezing, or other processing of whole blood collected from an individual.

[0046] Examples of blood sample diluents include, for example, a mixture of blood and buffer. Examples of buffers include, for example, Tris buffer, phosphate buffer, HEPES buffer, and ammonium acetate buffer.

[0047] In the diluent, organic solvents such as dimethylformamide and acetonitrile can be mixed. The final concentration of the organic solvent is, for example, 5–30 (v / v)%. Thus, during the preservation of blood samples collected from the subject, the degradation of the desired Ng peptide over time can be inhibited.

[0048] Furthermore, a surfactant can be mixed into the diluent. Examples of surfactants include neutral surfactants with a hydrophobic group having 7 to 15 carbon atoms. The concentration of the surfactant is, for example, 0.01 to 5 (v / v)%. This suppresses non-specific adsorption to the first conjugate or reduces ionization interference in mass analysis. Examples of such surfactants include: nonyl-β-D-maltose glycoside, nonyl-β-D-thiomaltose glycoside, decyl-β-D-maltose glycoside, and undecyl-β-D-maltose glycoside (UDM: n-Undecyl-β-D-maltoside), which have maltose in their hydrophilic portion; α-D-glucopyranosyl α-D-glucopyranosyl monodecanoate (trehalose C10), which have trehalose in their hydrophilic portion; and decyl-β-D-glucoside, which has glucose in its hydrophilic portion.

[0049] To normalize or standardize the measured values, it is preferable to further mix an internal standard into the blood sample. Stable isotope-labeled peptides are preferably examples of internal standards. The mixing amount of the internal standard relative to the plasma sample is, for example, more than 1 pM and less than 1 nM.

[0050] The first carrier only needs to be able to bind Ng peptides; for example, an antibody immobilization carrier can be cited. The antibody immobilized on the first carrier is an antibody with an antigen-binding site that can recognize Ng peptides (anti-Ng peptide antibody); for example, an immunoglobulin or fragment thereof with an antigen-binding site that can recognize Ng peptides can be cited. Examples of immunoglobulins include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgY, IgD, IgE, etc. Examples of immunoglobulin fragments include F(ab')2, F(ab'), F(ab), Fd, Fv, L chain, and H chain, etc. More specifically, this includes clones of NG2, NG7, EPR21152, and fragments thereof.

[0051] In the first cleaning step, after the first bonding step, the first bond is cleaned with the first cleaning solution.

[0052] The first washing solution is preferably a neutral buffer solution containing the aforementioned surfactant. This allows for the effective removal of unwanted components with high hydrophobicity (e.g., blood proteins, lipids, glycolipids, etc.).

[0053] The preferred cleaning method is to perform multiple cleaning cycles. For example, cleaning can be performed using a neutral buffer solution containing a surfactant, followed by cleaning using a neutral buffer solution without a surfactant. Any general cleaning method can be used, such as stirring the carrier in the cleaning solution or spraying the cleaning solution from a cleaning nozzle. After cleaning with these neutral buffer solutions, water-based cleaning can be performed further as needed.

[0054] In the first elution step, after the first washing step, the first conjugate is contacted with the first acidic solution. As a result, the Ng peptide dissociates from the first conjugate and elutes into the first acidic solution. The result is a first eluent containing the Ng peptide.

[0055] Examples of the first acidic solution include, for example, an acidic aqueous solution such as glycine buffer or hydrochloric acid (pH 0.5–3.5). The first acidic solution preferably contains the aforementioned surfactant. This allows for more reliable dissociation of the Ng peptide from the first conjugate, thereby improving detection sensitivity.

[0056] In the neutralization step, after the first elution step, the first eluent is mixed with a neutral buffer. This neutralizes the first eluent, yielding a purified solution containing the Ng peptide.

[0057] The neutral buffer solution used in the neutralization process is preferably containing the aforementioned surfactant. This suppresses non-specific adsorption of the second conjugate during the second binding process.

[0058] The resulting purified solution has a neutral pH, for example, pH 6.0 or higher, preferably 6.5 or higher, and furthermore, for example, pH 8.5 or lower, preferably 8.0 or lower. Therefore, the binding efficiency can be improved in the second binding step.

[0059] In the second binding step, after the neutralization step, the purified solution is contacted with the second carrier. Thereby, the Ng peptide in the purified solution binds to the second carrier, yielding the second conjugate.

[0060] The second carrier is preferably an antibody immobilization carrier; specifically, the same carrier as the antibody immobilization carrier illustrated in the first carrier can be cited.

[0061] In the second cleaning step, after the second bonding step, the second bond is cleaned with the second cleaning solution.

[0062] The second cleaning solution is preferably a neutral buffer solution containing the aforementioned surfactant. This allows for the effective removal, for example, of unwanted components with high hydrophobicity.

[0063] The cleaning method can be any known method; specifically, the same method as the cleaning method exemplified in the first cleaning step can be implemented.

[0064] In the second elution step, after the second washing step, the second conjugate is contacted with the second acidic solution. As a result, the Ng peptide dissociates from the second conjugate and elutes into the second acidic solution. The result is a second eluent containing the Ng peptide.

[0065] As the acidic aqueous solution constituting the second acidic solution, an example may be the same acidic aqueous solution as the first acidic solution illustrated in the first elution step, and hydrochloric acid may be a preferred example.

[0066] The second acidic solution is preferably containing a volatile organic solvent. This allows the Ng peptide to be efficiently dissociated from the second conjugate and eluted into the second acidic solution, thereby improving the recovery rate of the Ng peptide. Examples of volatile organic solvents include those that are mixed with water in any proportion, such as acetonitrile, methanol, ethanol, acetone, toluene, isopropanol, hexane, butanol, cyclohexane, ethylene glycol, benzene, chloroform, acetaldehyde, triethylamine, phenol, naphthalene, formaldehyde, tetrahydrofuran, and ethyl acetate.

[0067] The second acidic solution preferably further contains amino acids such as methionine. Therefore, during the period from when it is installed in the mass analysis device until the start of the analysis, the oxidation of Ng peptides can be reduced, and the detection sensitivity can be improved.

[0068] The second acidic solution preferably contains a protein of 9 kDa or more. Therefore, since the mass analysis sample contains the aforementioned protein, the protein acts as a proton acceptor during mass analysis, efficiently generating monovalent ions of Ng peptides. As a result, the detection sensitivity of Ng peptides can be significantly improved. The upper limit of the protein mass is, for example, 100 kDa or less, preferably 15 kDa or less. Examples of such proteins include bovine serum albumin (BSA), cytochromes, ovalbumin, and lysozyme.

[0069] (Quality Analysis Method)

[0070] After purification, the second eluent was subjected to mass analysis to detect Ng peptide.

[0071] Examples of ionization methods in mass analysis include MALDI (Matrix Assisted Laser Desorption / Ionization), ESI (Electrospray Ionization), and APCI (Atmospheric Pressure Chemical Ionization). From the viewpoint of being able to detect trace amounts of Ng peptides in plasma samples with high sensitivity, MALDI is a preferred example.

[0072] Examples of mass analysis methods include TOF-MS (Time-of-Flight Mass Analysis), IT-MS (Ion Trap Mass Analysis), IT-TOF-MS (Ion Trap-Time-of-Flight Mass Analysis), and FTICR-MS (Fourier Transform Ion Cyclotron Acceleration Mass Analysis), but these are not limited to these. When MALDI is used as the ionization method, these mass analysis methods are collectively referred to as MALDI-MS.

[0073] As a specific detection procedure based on MALDI-MS, for example, a matrix-containing solution is first added to a MALDI plate and allowed to dry, thereby preparing the matrix. Next, a second eluent is added to the matrix and allowed to dry, yielding a MALDI-MS sample. Then, the MALDI-MS sample is irradiated with a laser to ionize the Ng peptide, and the ionized peptide is detected by the detector of the aforementioned apparatus, as the mass spectrometer output.

[0074] Examples of matrix materials include α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, sinapic acid, and 3-aminoquinoline. Preferably, a matrix additive is used in conjunction with the matrix. Examples of matrix additives include phosphonic acid compounds such as methylene diphosphonic acid; ammonium salts, etc. Examples of solvents containing the matrix include acetonitrile, trifluoroacetic acid, methanol, ethanol, and water.

[0075] Thus, a mass spectrum of multiple Ng peptides contained in the blood sample is obtained, and the measured values ​​of the peak intensities (including peak areas) of the ion peaks corresponding to each of the multiple Ng peptides are obtained. Examples of such Ng peptides include those described later (refer to Tables 3 and 4). The measured value can be a relative intensity with the intensity of the reference peak set to 100%, or an intensity normalized by an internal standard (NI). Furthermore, if multiple measured values ​​(including normalized intensities) are obtained by dividing the second eluent into multiple portions (multiple traps), the average of these values ​​can also be used.

[0076] Furthermore, Ng peptides detected by mass spectrometry are sometimes detected as peaks of monovalent ions, and sometimes as peaks of monovalent ions and multivalent ions (divalent or higher). For example, both types of ions are detected in Ng43-75, etc. In this case, either method can be used as the measurement value for the biomarker. In the case of MALDI-MS, it is preferable to use the peak intensity detected at the monovalent ion peak for smaller Ng peptides with a mass less than 5000, and to use the peak intensity detected at the divalent ion peak for larger Ng peptides with a mass of 5000 or more. On the other hand, in the case of ESI (electrospray ionization), it is preferable to use the peak intensity detected at the multivalent ion peak.

[0077] 1-2. Application Process

[0078] In the application process, specific ratios are calculated using the measured values ​​of multiple Ng peptides obtained in the measurement process to determine the state of neurodegeneration. Specifically, multiple Ng peptide measurements are selected from those detected by mass spectrometry in the measurement process, and their ratios are calculated as indicators or scores used to determine the state of neurodegeneration.

[0079] In the first embodiment, "for determining the state of neurodegeneration" means that, in order to determine the state of neurodegeneration, a specific Ng peptide ratio (including normalized, averaged, etc. values) that serves as a biomarker can be used (the Ng ratio). For example, in addition to (1) using the Ng ratio directly as the indicator for determination, it also includes: (2) using the Ng ratio as a corrected or processed variable and using the corrected value or variable as the indicator for determination; (3) using the Ng ratio as its reciprocal and using that value as the indicator for determination; (4) using the Ng ratio to add, subtract, multiply, or divide the expected value or formula and using the value after addition, subtraction, multiplication, or division as the indicator for determination; (5) combinations thereof, etc.

[0080] The Ng ratio, as a biomarker, is at least one of the ratios described in (1) to (11) below. Furthermore, the Ng52-75 measurement is a measurement of Ng peptides such as Ng52-75 detected when measuring a blood sample, including the concentration, amount, or numerical value based on these of Ng52-75, which, in mass analysis, is, as described above, peak intensity (including normalized intensity, average value, etc.). The sequences of Ng peptides that are the objects of these ratios are shown in Table 1 below.

[0081] (1) The ratio of the measured value of Ng53-75 to the measured value of Ng52-75;

[0082] (2) The ratio of the measured value of Ng53-78 to the measured value of Ng51-75;

[0083] (3) The ratio of the measured value of Ng43-77 to the measured value of Ng43-75;

[0084] (4) The ratio of the measured value of Ng43-77 to the measured value of AcNg1-78ss;

[0085] (5) The ratio of the Ng43-77 measurement value to the sum of the AcNg1-75ss and AcNg1-78ss measurements;

[0086] (6) The ratio of the Ng43-77 measurement value to the sum of the AcCysNg1-75ss and AcCysNg1-78ss measurements;

[0087] (7) The ratio of the measured value of Ng43-77 to the total of the measured values ​​of Ng24-75 and Ng24-78;

[0088] (8) The ratio of the measured value of Ng43-78 to the measured value of Ng51-75;

[0089] (9) The ratio of the measured value of Ng24-78 to the measured value of AcCysNg1-75ss;

[0090] (10) The ratio of the total of Ng53-75 and Ng53-78 measurements to the Ng52-75 measurement;

[0091] (11) The ratio of the total of the Ng53-75 and Ng53-78 measurements to the Ng51-75 measurement.

[0092] [Table 1]

[0093]

[0094] The abbreviations in the table are as follows: Ac: Acetylation, Cys: Cysteinylation, Glu: Glutathionylation, ss: internal disulfide bond. Furthermore, acetylation occurs at the amino acid corresponding to position 1 of Ng1-78, glutathionylation occurs at the amino acid corresponding to position 3 or 9, and intermolecular disulfide bonds occur between the amino acids corresponding to positions 3 and 4, or between the amino acids corresponding to positions 4 and 9 (see Non-Patent Literature 5).

[0095] These biomarkers can be used individually or in combination. From the viewpoint of having high resolution for both Alzheimer's disease and non-Alzheimer's dementia, it is preferable to have at least one value from (1) to (2), at least one value from (3) to (7), or at least one value from (10) to (11).

[0096] Next, the Ng ratio, which serves as a biomarker, is used to determine the state of neurodegeneration.

[0097] Specifically, the Ng ratio of the aforementioned biomarkers in a blood sample collected from the subject is compared with a predetermined value. Then, if the Ng ratio of the biomarker is higher or lower than the predetermined value, it is determined that the subject's brain has undergone neurodegeneration, and the subject suffers from dementia. Examples of comparison schemes include: 1) comparing the Ng ratio in a blood sample collected from the subject with a pre-set baseline value indicating normal cognitive function; 2) collecting the Ng ratios of multiple blood samples collected from the subject over time (e.g., every few months) and comparing these multiple Ng ratios; 3) combinations of these, etc. Furthermore, the baseline value in 1) can be appropriately set based on the Ng ratios of blood samples collected in advance from multiple cognitively normal individuals and / or dementia patients.

[0098] In particular, the Ng ratios mentioned in (1) to (11) above are increased in the blood of subjects with neurodegeneration. Therefore, in scheme 1), if the Ng ratio in the subject is higher than the baseline value for normal cognitive function, it is judged that the subject's brain has undergone neurodegeneration. In scheme 2), if the Ng ratio measured on a specific date is higher than the Ng ratio measured on a previous date, it is judged that the subject's brain has undergone neurodegeneration or that neurodegeneration is progressing. On the other hand, in the opposite case, that is, in schemes 1) and 2), if the former measurement value decreases, it is judged that the neurodegeneration of the subject's brain is suppressed.

[0099] In this way, the method for estimating the Ng ratio using biomarkers in the first embodiment involves detecting and using Ng peptides in a blood sample. The method measures the blood sample collected from the subject, selects the measured values ​​of multiple Ng peptides from the measurement results, calculates and uses these ratios, thereby easily estimating the neurodegenerative state of the brain, i.e., whether functional loss of brain nerve cells has occurred. For example, it is easy to predict the presence or progression of synaptic dysfunction, synaptic loss, etc. Therefore, this method can be used as information for diagnosing whether a subject suffers from dementia caused by neurodegeneration. Specifically, as a type of dementia caused by neurodegeneration, in addition to the representative Alzheimer's disease, it can also be used as information for diagnosing dementia including non-Alzheimer's dementia types (Lewy body type, frontotemporal type, etc.). Furthermore, the mechanism by which dementia develops is caused by neurodegeneration. Since neurodegeneration occurs earlier than the onset of the disease, it can also be used as information for predicting the onset of these dementias and diagnosing the risk of developing the disease.

[0100] In particular, this inference method allows for easy inference of the neurodegenerative state of the brain from thawed and preserved blood samples. Blood samples are in a liquid state at the time of collection or during centrifugation, existing at room temperature or above 0°C. Furthermore, they are typically cryopreserved to maintain freshness. The time from blood collection to cryopreservation varies depending on the medical institution and blood collection location. Moreover, blood samples may include those that have been thawed to a liquid state, stored at room temperature or above 0°C for several hours, and then refrozen. In such blood samples, the amount of Ng peptides increases or decreases during the liquid state before cryopreservation, and changes occur at the time of blood collection. This change can be attributed to the activity of enzymes in the blood during the liquid state, producing decomposition products along with the Ng peptides. Therefore, it becomes impossible to accurately determine the state of the blood at the time of collection. In contrast, in this inference method, by setting the biomarker to the aforementioned specific Ng peptide ratio, that is, by setting it to the relative value of a variable specific Ng peptide measurement to a variable specific other Ng peptide measurement, the influence of the variation of Ng peptide in the liquid state can be reduced, and the neurodegenerative state of the brain can be accurately inferred.

[0101] Furthermore, this inferential method can also evaluate the effectiveness of medical interventions. For example, if a patient is diagnosed with neurodegeneration, a medical intervention is initiated, and then the inferential method is repeated to assess the state of neurodegeneration. Upon re-implementation, if the inferential method indicates that the progression of neurodegeneration has stopped, been inhibited, delayed, or is absent, the medical intervention can be evaluated as effective. Conversely, if the inferential method indicates that neurodegeneration is progressing, the medical intervention can be evaluated as ineffective. Moreover, this method can also be applied to situations where, even without medical intervention, the progression of neurodegeneration is being monitored after a diagnosis of neurodegeneration.

[0102] 1-3. Variations

[0103] 1) In the above embodiment, the Ng ratios (1) to (11) are used to determine the neurodegenerative state. However, for example, multiple measurements can be combined and a variable can be calculated using multivariate analysis, and this variable can be used to determine the neurodegenerative state. That is, two measurements can be selected from the group consisting of (1) to (11), and these selected measurements can be calculated as variables using multivariate analysis, and this variable can be used to determine the neurodegenerative state. As a result, the AUC (Area Under Curve) of ROC analysis is further improved, enabling more accurate determination.

[0104] As a multivariate analysis, well-known mathematical methods can be used, such as discriminant analysis (linear discriminant method, quadratic discriminant method, normalized discriminant method, etc.), multiple regression analysis, kernel methods, principal component analysis, partial least squares regression, logistic regression, Z-score, etc.

[0105] 2) In the above embodiments, a quality analysis method is implemented as the measurement step, but the measurement method is not limited; for example, a sandwich immunoassay can also be implemented. In this method, by applying a sandwich method to the N-terminal specific antibody and the antibody recognizing the C-terminal region of each of the above-mentioned Ng peptides, the Ng peptides that serve as the above-mentioned biomarkers can be measured. Furthermore, the Ng peptides with Ng53 as the N-terminus present in blood samples are mainly Ng53-75 and Ng53-78, while others are trace amounts that cannot be detected by quality analysis and have almost no effect on the combined value of Ng53-75 and Ng53-78. Therefore, by applying a sandwich method to the N-terminal specific antibody of Ng53-75 and the antibody recognizing the C-terminal region of N53, the combined value of Ng53-75 and Ng53-78 can be measured.

[0106] In addition, the measurement values ​​of each Ng peptide are determined appropriately according to the measurement method, such as concentration, absorbance, etc.

[0107] 2. Plan

[0108] Those skilled in the art should understand that the above-described exemplary embodiments are specific examples of the following scheme.

[0109] (Item 1) A method for inferring the neurodegenerative state of the brain involved in a scheme may include: a measurement step, measuring a blood sample collected from a subject to obtain measurement values ​​of multiple neurogranule protein-related peptides; and a use step, using the measurement values ​​of the multiple neurogranule protein-related peptides to calculate the value of at least one of the following (1) to (11), and using the value of the ratio to determine the neurodegenerative state.

[0110] (1) The ratio of the measured value of Ng53-75 to the measured value of Ng52-75;

[0111] (2) The ratio of the measured value of Ng53-78 to the measured value of Ng51-75;

[0112] (3) The ratio of the measured value of Ng43-77 to the measured value of Ng43-75;

[0113] (4) The ratio of the measured value of Ng43-77 to the measured value of AcNg1-78ss;

[0114] (5) The ratio of the Ng43-77 measurement value to the sum of the AcNg1-75ss and AcNg1-78ss measurements;

[0115] (6) The ratio of the Ng43-77 measurement value to the sum of the AcCysNg1-75ss and AcCysNg1-78ss measurements;

[0116] (7) The ratio of the measured value of Ng43-77 to the total of the measured values ​​of Ng24-75 and Ng24-78;

[0117] (8) The ratio of the measured value of Ng43-78 to the measured value of Ng51-75;

[0118] (9) The ratio of the measured value of Ng24-78 to the measured value of AcCysNg1-75ss;

[0119] (10) The ratio of the total of Ng53-75 and Ng53-78 measurements to the Ng52-75 measurement;

[0120] (11) The ratio of the total of the Ng53-75 and Ng53-78 measurements to the Ng51-75 measurement.

[0121] (Item 2) In the deduction method of Item 1, it may be that, during the use process, if the value of the ratio is higher than a predetermined value, the subject's brain is judged to have undergone neurodegeneration.

[0122] (Item 3) In the estimation method of Item 1, it may be that, during the use process, the value of the ratio is compared with a pre-set benchmark value for normal cognitive function.

[0123] (Item 4) In the estimation method of Item 1, it may be that the measurement process is performed multiple times over time, and the use process is a process of comparing the values ​​of multiple ratios obtained and calculated through multiple implementations.

[0124] (Item 5) In any of the deduction methods in items 1 to 4, it may be that, in the usage step, two ratio values ​​are selected from the ratios obtained by using the measurements of the plurality of neuroparticle protein-related peptides, and the variables are calculated using multivariate analysis and used to determine the neurodegenerative state.

[0125] (Item 6) In any of the speculation methods in items 1 to 5, it may be that, in the use process, at least one of the ratios in (1) to (2) is used.

[0126] (Item 7) In any of the speculation methods in items 1 to 5, it may be that, in the use process, at least one of the ratios in (3) to (7) is used.

[0127] (Item 8) In any of the speculation methods in items 1 to 5, it may be that, in the use process, at least one of the ratios in (10) to (11) is used.

[0128] (Item 9) In any of the deduction methods in items 1 to 8, it may be that, in the measurement process, a mass analysis method is performed on the blood sample to obtain a measured value of the peak intensity of the plurality of neuroparticle protein-related peptides.

[0129] (Item 10) In the speculative method of Item 9, it may be that the blood sample is subjected to affinity purification before the quality analysis method.

[0130] (Item 11) A method for detecting neurogranule protein-related peptides, comprising:

[0131] The procedure of detecting neurogranule protein-related peptides in a blood sample from at least one combination selected from the group consisting of (1) to (11) below.

[0132] (1) The combination of Ng52-75 and Ng53-75;

[0133] (2) The combination of Ng51-75 and Ng53-78;

[0134] (3) The combination of Ng43-75 and Ng43-77;

[0135] (4) The combination of AcNg1-78ss and Ng43-77;

[0136] (5) Combinations of AcNg1-75ss, AcNg1-78ss and Ng43-77;

[0137] (6) Combinations of AcCysNg1-75ss, AcCysNg1-78ss and Ng43-77;

[0138] (7) Combinations of Ng24-75, Ng24-78 and Ng43-77;

[0139] (8) Combination of Ng51-75 and Ng43-78;

[0140] (9) The combination of AcCysNg1-75ss and Ng24-78;

[0141] (10) Combinations of Ng52-75, Ng53-75 and Ng53-78;

[0142] (11) Combinations of Ng51-75, Ng53-75 and Ng53-78.

[0143] Example

[0144] Next, the present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0145] 1. Plasma sample

[0146] At the National Center for Longevity Medicine, plasma samples from 161 cases were obtained, along with clinical diagnostic information including amyloid PET, FDG-PET, MRI, and MMSE (Mini Mental State Examination). Detailed information is shown in Table 2.

[0147] [Table 2]

[0148]

[0149] The subjects were individuals diagnosed with cognitively normal function (CN), mild cognitive impairment (MCI), Alzheimer's disease (AD), and dementia other than Alzheimer's disease (nonAD). The groups summing the AD and nonAD cases were designated as the dementia group.

[0150] FDG-PET diagnostic use will18 Quantitative FDG-PET scores based on F-FDG-PET scans (refer to Herholz K et al.; Evaluation of a calibrated (18)F-FDG PET score as a biomarker for progression in Alzheimer disease and mild cognitive impairment. J Nucl Med. 2011 Aug; 52(8): 1218-26.).

[0151] MRI diagnosis used a morphometry-based assessment of hippocampal atrophy using VSRAD (see Sone D et al.; Japanese-Alzheimer's Disease Neuroimaging Initiative. Voxel-based Specific Regional Analysis System for Alzheimer's Disease (VSRAD) on 3-tesla Normal Database: Diagnostic Accuracy in Two Independent Cohorts with Early Alzheimer's Disease. AgiNg Dis. 2018 Aug 1; 9(4): 755-760.).

[0152] To determine whether Aβ accumulation in the brain is positive or negative, PiB-PET images of the subject's brain were acquired. Subjects were assessed visually by a nuclear medicine specialist, and those with greater or equal PiB accumulation in the cerebral cortex than in nonspecific white matter were considered positive. Subjects with only nonspecific accumulation in white matter and almost no accumulation in the cortex were considered negative. The PiB accumulation mean (SUVR: Standard Uptake Value Ratio) was used to quantify cortical PiB accumulation, and the cerebral accumulation ratio was calculated using the cerebellum as a baseline. Furthermore, AD cases confirmed as Aβ-positive in amyloid PET were designated as AD_Aβ+ (N=39), and nonAD cases confirmed as Aβ-negative in amyloid PET were designated as nonAD_Aβ- (N=41).

[0153] <Experimental Example 1>

[0154] 1. Measurement of Ng peptide in the control sample

[0155] [Preparation of blood samples]

[0156] DMSO (dimethyl sulfoxide) was mixed into a first buffer containing surfactant (0.1% n-undecyl-β-D-maltose (UDM), 800 mM MClNAc, 100 mM Tris-HCl, 300 mM NaCl; pH 7.4) to prepare a DMSO concentration of 20% (v / v). Next, as internal standards, stable isotope-labeled Ng53-75 (SIL-Ng53-75), Ng45-75 (SIL-Ng45-75), and Ng24-75 (SIL-Ng24-75) were mixed at 30 pM, 50 pM, and 50 pM, respectively, to prepare a buffer containing internal standards. Then, 200 μL of human plasma (control sample) was mixed into 200 μL of the buffer containing internal standards and incubated on ice for 5–60 minutes. This prepared the plasma sample. In addition, in SIL-Ng45-75 and SIL-Ng24-75, the carbon and nitrogen atoms of some amino acids of Pro, Val, and Ala are respectively... 13 C and 15 N is replaced.

[0157] [purification]

[0158] (First bonding process, first cleaning process, first elution process)

[0159] Cloner NG2 (BioLegend) of an anti-Ng antibody (IgG1) with epitopes at residues 52-63 of human neurogranule protein (Ng) was prepared. 5.5 mg of magnetic beads (Dynabeads M-270Epoxy) were reacted with 100 μg of anti-Ng antibody in immobilization buffer (0.1 M phosphate buffer containing 1.5 M ammonium sulfate; pH 7.4) at 37°C for 16–24 hours. This produced antibody beads that served as the antibody immobilization carrier.

[0160] The plasma sample was mixed with the antibody beads described above and incubated at 4°C for 1 hour. Then, the antibody beads were washed once with 100 μL of the first washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4) and once with 50 μL of 50 mM ammonium acetate buffer. Next, the antibody beads were contacted with the first acidic solution (0.05% UDM, 50 mM glycine buffer; pH 2.8) to elute the Ng peptide into the first acidic solution. This yielded the first eluent containing the Ng peptide.

[0161] (Neutralization process)

[0162] The first elution buffer was mixed with a neutral buffer (0.1% UDM, 800 mM GlcNAc, 300 mM Tris-HCl, 300 mM NaCl; pH 7.4) to obtain the purified solution.

[0163] (Second bonding process, second cleaning process, second elution process)

[0164] The purified solution was mixed with antibody beads and incubated at 4°C for 1 hour. Then, the antibody beads were washed twice with 50 μL of second washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4), once with 50 μL of 50 mM ammonium acetate buffer, and once with 30 μL of water. Next, the antibody beads were contacted with a second acidic solution (70% (v / v) acetonitrile aqueous solution containing 5 mM hydrochloric acid and 0.1 mM methionine, 50 nM BSA) to elute the Ng peptide into the second acidic solution. This yielded the second eluent containing the Ng peptide.

[0165] [Quality Analysis]

[0166] The MALDI-TOF MS system used was an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) device. α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix for linear TOF, methylene diphosphonic acid (MDPNA) was used as the matrix additive, and acetonitrile was used as the solvent to prepare a 2 mg / mL CHCA / 0.2% (w / v) MDPNA matrix solution. 0.5 μL of the matrix solution was added dropwise to each of the four traps of a MALDI plate (μFocus MALDI plate 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ)). After drying, 1 μL of the second elution buffer was added dropwise to each trap, and then dried.

[0167] Next, MALDI-TOF MS was used to detect Ng peptides. Mass spectrometry data were acquired via linear TOF in positive ion mode as a set condition. For each trap, 400 points were accumulated, totaling 16,000 shots. The m / z values ​​of the linear TOF were expressed as the average mass of the peaks. The m / z values ​​were calibrated using human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, bovine insulin, and cytochrome C as external standards.

[0168] Tables 3 and 4 show the Ng peptides detected in mass spectrometry. * in the tables indicates divalent ion peaks.

[0169] [Table 3]

[0170] Peptide Name Sequence Theoretical Average m / z Number <![CDATA[Ng43-75 * ]]> RKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 1493.2 13 <![CDATA[Ng43-78 * ]]> RKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGPSGD 1622.8 5 Ng53-75 RKGPGPGG PGGAGVARGG AGGGP 1845.0 1 SIL-Ng53-75 RKGPGPGG PGGAGVARGG AGGGP 1877.1 1 Ng52-75 GRKGPGPGG PGGAGVARGG AGGGP 1902.1 6 Ng51-75 RGRKGPGPGG PGGAGVARGG AGGGP 2058.3 7 Ng53-78 RKGPGPGG PGGAGVARGG AGGGPSGD 2104.3 2 Ng50-75 E RGRKGPGPGG PGGAGVARGG AGGGP 2187.4 8 Ng43-65 RKKIKSGE RGRKGPGPGG PGGAG 2205.5 9 Ng48-75 SGE RGRKGPGPGG PGGAGVARGG AGGGP 2331.5 3 Ng42-66 ARKKIKSGE RGRKGPGPGG PGGAGV 2375.8 10 Ng50-78 E RGRKGPGPGG PGGAGVARGG AGGGPSGD 2446.6 11 Ng45-75 KIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 2701.0 12 SIL-Ng45-75 KIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 2733.1 12 Ng44-75 KKIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 2829.2 4 Ng43-75 RKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 2985.4 13 Ng43-77 RKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGPSG 3129.5 14 Ng42-77 ARKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGPSG 3200.6 15 Ng43-78 RKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGPSGD 3244.6 5 Ng42-78 ARKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGPSGD 3315.7 16 Ng40-75 H MARKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 3324.8 17 Ng37-70 FRGH MARKKIKSGE RGRKGPGPGG PGGAGVARGG 3345.9 18 Ng38-75 RGH MARKKIKSGE RGRKGPGPGG PGGAGVARGG AGGGP 3538.1 19

[0171] [Table 4]

[0172]

[0173] [Measurement values ​​of Ng peptides]

[0174] For a single subject, the peak intensity (NI) of each Ng peptide, normalized by the internal standard peptide (SIL-Ng53-75, SIL-Ng45-75, or SIL-Ng24-75), is calculated in each of the four spectra. The average of the four peak intensities is taken as the measured value of the Ng peptide for each subject. Peak intensities (NI) that do not reach the detection limit (S / N < 3) are excluded. The maximum number of data points used for averaging peak intensities (NI) is four; however, if the number of data points is less than three, the peak intensity (NI) is considered undetectable and is defined as 0.

[0175] In addition, the reproducibility of peak intensity (NI) was evaluated using commercially available plasma supplemented with Ng peptide and recombinant Ng (recombinant neurogranulin). The results showed that, over 4 days of daily reproducibility, Ng53-75 had a CV of 2.3%, Ng48-75 a CV of 7.8%, Ng45-75 a CV of 6.9%, Ng43-75 a CV of 5.2%, and Ng24-75 a CV of 3.7%. The divalent ion of recombinant neurogranulin had a CV of 9.8%, and the monovalent ion of recombinant neurogranulin had a CV of 15.7%. Furthermore, regarding simultaneous reproducibility, four batches were evaluated: Ng53-75 (4.9-9.7% CV), Ng48-75 (3.4-6.2% CV), Ng45-75 (2.1-5.5% CV), Ng43-75 (4.1-11.2% CV), and Ng24-75 (1.8-7.1% CV). The divalent ion of recombinant neurogranules showed a CV of 4.6-14.5%, and the monovalent ion of recombinant neurogranules showed a CV of 3.4-10.3%. This method offers analytical precision below 20% CV, indicating reliable reproducibility.

[0176] Furthermore, for Ng peptides that detected both monovalent and divalent ions, the same Ng peptide was measured. Therefore, for smaller Ng peptides with a mass less than 5000, the monovalent ion was used as the analytical target, while for larger Ng peptides with a mass greater than 5000, the divalent ion was used as the analytical target.

[0177] 2. Measurement of Ng peptides in samples stored at room temperature

[0178] The human plasma (control sample) was thawed, left at room temperature for approximately 3 hours, and then refrozen. Using this human plasma (RT3h-FT1 sample), the Ng peptide values ​​were measured in the same manner as described above.

[0179] 3. Variation rate of Ng peptides in plasma

[0180] Calculate the ratio (rate of change) of the measured values ​​of each Ng peptide in the RT3h-FT sample relative to the measured values ​​of each Ng peptide in the control sample, and then plot it in...Figure 1 As shown. By Figure 1 It can be seen that the amount of Ng peptide in plasma increases or decreases when thawed, stored at room temperature, or refrozen.

[0181] 4. Calculation of the Ng peptide / Ng peptide ratio

[0182] Through the Figure 1 The 26 groups of Ng peptides recorded were each divided by the 26 groups of Ng peptides, resulting in a total of 650 Ng peptide / Ng peptide ratios (hereinafter referred to as Ng ratios). Among them, 187 Ng ratios (28.8%) exhibited a good low variability rate with a variability rate within ±20%, and 84 Ng ratios (12.9%) exhibited a low variability rate with a variability rate of 20%–30% or -20%–-30%. Therefore, among these 271 Ng ratios, the variability caused by thawing, room temperature storage, and refrozening was suppressed, making them potential candidates for accurate biomarkers that account for the increase or decrease of Ng peptides during thawing, room temperature storage, and refrozening.

[0183] [Validation of Ng ratio as a biomarker]

[0184] AD_Aβ+ or nonAD_Aβ- is diagnosed as dementia, a condition characterized by cognitive decline, and therefore originates from the plasma of subjects who have already undergone neurodegeneration of the brain. Thus, an increase or decrease in AD_Aβ+ or nonAD_Aβ- compared to the CN group can be used as a biomarker reflecting changes in brain neurodegeneration. Therefore, median values ​​were calculated for the CN, MCI, AD_Aβ+, and nonAD_Aβ- groups out of 407 Ng ratios.

[0185] The result of screening 271 Ng ratios and selecting those that meet either screening criterion (1) or (2) is that 101 ratios exist. Furthermore, the result of screening 161 subjects for Ng ratios that account for more than 80% of the subjects (more than 129 subjects) is that 89 ratios exist.

[0186] [Screening Criteria]

[0187] (1) The above variation rate is within ±20%, and the median value of AD_Aβ+ or nonAD_Aβ- relative to CN is more than 1.5 times or less than 0.666 times.

[0188] (2) The above-mentioned rate of change exceeds ±20% but is within ±30%, and the median value of AD_Aβ+ or nonAD_Aβ- relative to CN is more than 2 times or less than 0.5 times.

[0189] Next, to evaluate the ability to detect AD_Aβ+ or nonAD_Aβ-, ROC analysis was performed on these 89 Ng ratios. The results showed that 12 Ng ratios exhibited very high precision, with AUC values ​​above 0.8. Of these, one ratio, which was in a reciprocal relationship (only the numerator and denominator were in opposite positions), showed only an inverse correlation with the pathological state and was therefore treated as a single biomarker, leaving 11 Ng ratios remaining.

[0190] Regarding these 16 Ng ratios, Table 5 shows the median values ​​of the CN group, MCI group, AD_Aβ+ group, and nonAD_Aβ- group; Table 6 shows the differences between the median values ​​of the MCI group, AD_Aβ+ group, and nonAD_Aβ- group and the CN group; Table 7 shows the test for the differences in median values; and Table 8 shows the AUC.

[0191] [Table 5]

[0192]

[0193] [Table 6]

[0194]

[0195] [Table 7]

[0196]

[0197] [Table 8]

[0198]

[0199] exist Figures 3-8 This study shows the association between 11 Ng ratio biomarkers and FDG-PET, VSRAD, and MMSE. MMSE is a score evaluating cognitive function; FDG-PET assesses brain glucose metabolism to indicate decreased function of brain neurons and synapses; and VSRAD, based on MRI, shows brain atrophy caused by the loss of neurons in the medial temporal lobe. These are used as indicators of neurodegeneration in the brain. Higher FDG-PET and VSRAD scores indicate more advanced neurodegeneration, as does a lower MMSE score.

[0200] In the association between various Ng ratio biomarkers and FDG-PET, VSRAD, and MMSE, cases showing high values ​​in FDG-PET and VSRAD also showed a tendency for a relatively higher Ng ratio. Figures 3-5 , Figures 9-11 Even in cases where MMSE values ​​were low, the Ng peptide ratio similarly showed a tendency to be relatively increased. Figures 6-8These results indicate that these Ng ratios capture changes in brain neurodegeneration that can be detected by FDG-PET, VSRAD, and MMSE. Furthermore, the Ng ratio shows a high tendency even for dementia cases that cannot be captured by FDG-PET, VSRAD, and MMSE. This suggests that it also captures brain neurodegeneration in dementia cases missed by FDG-PET, VSRAD, and MMSE.

[0201] In addition, the possibility that the difference between the CN group and AD_Aβ+ or nonAD_Aβ- was due to the difference between Aβ positivity and Aβ negativity as pathological features of AD was also considered. However, the difference in median values ​​between the Aβ-positive and Aβ-negative groups within the CN group, the difference in median values ​​between the Aβ-positive and Aβ-negative groups within the dementia case group, and the difference in median values ​​between the AD_Aβ+ group and the nonAD_Aβ- group were not statistically significant (Table 9).

[0202] [Table 9]

[0203]

[0204] In summary, the 11 Ng peptide ratio biomarkers do not only reflect changes in AD pathology, but also reflect dementia caused by neurodegeneration of the brain (including synaptic disorders).

[0205] <Experimental Example 2>

[0206] [Multivariate Analysis of Ng Ratio as a Biomarker]

[0207] Eleven Ng peptide biomarkers were used to calculate the predicted probability of dementia based on logistic regression analysis, and ROC analysis was performed. The results showed that CN vs AD_Aβ+ and CN vs nonAD_Aβ- yielded higher AUCs than any of the 11 Ng peptide biomarkers (Table 10).

[0208] The mean and standard deviation of Ng53-78 / Ng51-75 and [Ng53-75+Ng53-78] / Ng51-75 from 161 subjects were normalized to calculate the z-score. The composite z-score was calculated by averaging Ng53-78 / Ng51-75 and [Ng53-75+Ng53-78] / Ng51-75 (Table 11). The results showed that CN vs AD_Aβ+, CN vs nonAD_Aβ-, and CN vs MCI yielded AUCs higher than any of the 11 Ng biomarkers (Table 11).

[0209] The results above indicate an improved ability to detect neurodegeneration in the brain through multivariate analysis.

[0210] [Table 10]

[0211]

[0212] [Table 11]

[0213]

[0214] <Comparison with the markers in non-patent documents 5-7>

[0215] Non-patent documents 5-7 disclose Ng molecules shown in Table 12 as biomarkers. In these documents, a comparison was made between two groups of control groups (healthy individuals) and AD, and p-values ​​were calculated. These are shown in Table 12. Generally, the larger the difference between the two groups, the lower the p-value. Among the Ng molecules shown in Table 12, even the lowest p-value is 0.002 for Ng48-76. On the other hand, in the inter-group differences of the above 11 Ng biomarkers NC vs AD_Aβ+ corresponding to the difference between the control group (healthy individuals) and AD, p < 0.0001 is shown (Table 7), indicating that the difference between the two groups is greater than that of the Ng molecules shown in Table 12.

[0216] Furthermore, as detection methods, the Mann-Whitney U test was used in Non-Patent Literature 5, the Mann-Whitney U test was used in Non-Patent Literature 6, and the Wilcoxon rank-sum test was used for the aforementioned 11 Ng markers. The Mann-Whitney U test and the Wilcoxon rank-sum test are equivalent; therefore, these test methods were used for the markers in the aforementioned Non-Patent Literature and the aforementioned 11 markers. Additionally, in Table 12, "Total FL" is the value of Non-Patent Literature 5. Figure 7 The sum of the three peaks of Ng1-78 with slightly different post-translational modifications, as shown in c.

[0217] [Table 12]

[0218]

[0219] Of the Ng molecules shown in Table 12, apart from Ng53-75 and Ng53-78, in the detection of Ng molecules in the blood sample shown in Experimental Example 1, if they were present in the sample at a concentration exceeding the detection limit of this measurement method, they should have been detected, but were not (see Tables 3 and 4). Therefore, it can be said that molecular markers other than Ng53-75 and Ng53-78 disclosed in Non-Patent Documents 5-7 are not effective as markers in blood samples.

Claims

1. A method for inferring the neurodegenerative state of the brain, characterized in that, have: The measurement process involves measuring blood samples collected from the subject to obtain the measured values ​​of multiple neurogranule protein-related peptides. In the process of using the measured values ​​of the plurality of neurogranule protein-related peptides, at least one of the ratios in (1) to (11) below is calculated, and the value of the ratio is used to determine the neurodegenerative state. (1) The ratio of the measured value of Ng53-75 to the measured value of Ng52-75; (2) The ratio of the measured value of Ng53-78 to the measured value of Ng51-75; (3) The ratio of the measured value of Ng43-77 to the measured value of Ng43-75; (4) The ratio of the measured value of Ng43-77 to the measured value of AcNg1-78ss; (5) The ratio of the Ng43-77 measurement value to the sum of the AcNg1-75ss and AcNg1-78ss measurements; (6) The ratio of the Ng43-77 measurement value to the sum of the AcCysNg1-75ss and AcCysNg1-78ss measurements; (7) The ratio of the measured value of Ng43-77 to the total of the measured values ​​of Ng24-75 and Ng24-78; (8) The ratio of the measured value of Ng43-78 to the measured value of Ng51-75; (9) The ratio of the measured value of Ng24-78 to the measured value of AcCysNg1-75ss; (10) The ratio of the total of Ng53-75 and Ng53-78 measurements to the Ng52-75 measurement; (11) The ratio of the total of the Ng53-75 and Ng53-78 measurements to the Ng51-75 measurement.

2. The inference method as described in claim 1, characterized in that, In the process of use, if the value of the ratio is higher than a specified value, it is determined that the subject's brain has undergone neurodegeneration.

3. The inference method as described in claim 1, characterized in that, In the usage process, the value of the ratio is compared with a preset benchmark value for normal cognitive function.

4. The inference method as described in claim 1, characterized in that, The measurement process is performed multiple times over time. The process of using the method is a process of comparing the values ​​of multiple ratios obtained and calculated through multiple implementations.

5. The inference method as described in claim 1, characterized in that, In the usage step, two ratios are selected from the ratios obtained by using the measurements of the multiple neurogranule protein-related peptides, and the variables are calculated using multivariate analysis. The variables are used to determine the state of neurodegeneration.

6. The method for inferring the neurodegenerative state of the brain as described in claim 1, characterized in that, In the process of use, at least one of the ratios in (1) to (2) is used.

7. The method for inferring the neurodegenerative state of the brain as described in claim 1, characterized in that, In the process of use, at least one of the ratios in (3) to (7) is used.

8. The method for inferring the neurodegenerative state of the brain as described in claim 1, characterized in that, In the process of use, at least one of the ratios in (10) to (11) is used.

9. The inference method as described in claim 1, characterized in that, In the measurement process, a mass analysis method is performed on the blood sample to obtain measured values ​​of the peak intensities of the multiple neuroparticle protein-related peptides.

10. The inference method as described in claim 9, characterized in that, In the measurement process, the blood sample undergoes affinity purification prior to the mass analysis method.

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