WO2022070798A1 - Combined markers for differentiating pathological conditions of alzheimer's disease and method of differentiating pathological conditions of alzheimer's disease using same - Google Patents

Abstract

The present invention addresses the problem of developing biomarkers, by which the occurrence or absence of AD and the onset risk thereof can be specifically and highly sensitively differentiated, and thus providing a kit for detecting the biomarkers and a method of differentiating the pathological conditions of AD using the same.　The combined markers according to the present invention enable highly accurate differentiation of the pathological conditions of AD in a subject, namely, the occurrence or absence of AD or MCI and the onset risk thereof.

Description

Combination marker for differentiating Alzheimer's disease and method for differentiating Alzheimer's disease using it

The present invention presents Alzheimer's disease and a combination biomarker for early identification of cognitive disorders that may progress to Alzheimer's disease from other central nervous system diseases, a kit for detecting them, and an Alzheimer's disease using the combination biomarker. Regarding the method of distinguishing the pathological condition.

Alzheimer's disease (hereinafter often abbreviated as "AD" in the present specification) is a type of irreversible progressive central nervous system disease, and is a cognitive dysfunction (dementia) accompanied by memory impairment and thinking disorder. , Behavioral disorders, or personality changes. It is the most common dementia disease and accounts for about 60-80% of all dementia patients. It generally develops in the elderly aged 65 and over, but some also develop in the age of 64 and under, and is called juvenile Alzheimer's disease.

The number of dementia patients worldwide is estimated to be more than 50 million as of 2019, and is expected to reach 150 million by 2050. Of these, about 70% are considered to be AD patients. Especially in developed countries, the economic or mental burden on the national government and patients is becoming more serious due to the increase in medical expenses and long-term care problems due to the increase in AD patients, and it is becoming a big social problem. In the brains of AD patients, the hydrophobic peptide amyloid β protein (Aβ) begins to aggregate and accumulate in the brain, and then the microtubule protein tau protein is hyperphosphorylated and fibrotic, followed by nerve cells. Is destroyed and the brain is atrophic. Familial (hereditary) AD develops early and becomes juvenile AD due to mutations in the amyloid precursor protein (APP) gene and presenilin 1 (PSEN1) gene involved in these metabolisms.

Currently, based on the pathogenic mechanism of AD, many AD therapeutic agents that inhibit the pathogenic process are being developed. However, most of them have not yet been shown to be sufficiently effective in clinical trials. It is hypothesized that this is not because the investigational drug is ineffective, but because the study group is not appropriate. The target group for which clinical trials have been conducted in the past was late-stage patients with advanced brain lesions due to AD and massive neuronal cell death. However, even if treatment is started at this stage, recovery of nerve function cannot be expected. That is, it is considered that an effective therapeutic effect could not be obtained because the administration time of the therapeutic agent was too late (Non-Patent Document 1). In 2021, a monoclonal antibody against soluble Aβ aggregates was approved by the FDA for the first time in the world as a treatment for AD. However, although this therapeutic agent improves amyloid pathology, it does not improve cognitive symptoms (Non-Patent Document 2). This means that the effect of this therapeutic agent is limited to suppressing the progression of AD pathology. Therefore, if the AD therapeutic agent can be administered at an appropriate timing, that is, at an early stage in which nerve cell death can be avoided, its effectiveness can be expected. For that purpose, it is necessary to distinguish the AD pathological condition with higher accuracy.

In general, patients with suspected dementia who have visited the outpatient department for forgetfulness have AD, mild cognitive impairment (in the present specification) based on tests such as the mini-mental state examination (MMSE). It is divided into three groups: (often abbreviated as "MCI") and a group that does not meet the diagnostic criteria for MCI and AD (Normal Control: often abbreviated as "NC" in the present specification). Studies have shown that some NC patients may transition to MCI, which is dementia in the future, and some MCI may transition to more severe AD in the future, but dementia scores alone are not enough. It was a poorly objective diagnostic method, and it was not possible to judge the transition to aggravation.

As a method for objectively and earlyly differentiating AD pathological conditions, a method using a causative factor of AD as a marker for AD diagnosis is known. Such biomarkers include tau protein, overly phosphorylated tau (p-tau) protein, amyloid β42 (Aβ42) peptide, amyloid β40 (Aβ40) peptide or cytokines in inflammatory diseases (Non-Patent Document 3). In addition, a transferase sugar protein having a mannose non-reducing terminal sugar chain (terminal Man-Tf protein) (Patent Document 1) is known.

However, tau protein is not an AD-specific biomarker because it also increases in dementia such as frontotemporal dementia and progressive supranuclear palsy. On the other hand, the p-tau protein is an excellent marker for AD diagnosis, but the appearance of this protein means the death of nerve cells, and although it is appropriate for early diagnosis of AD in which nerve cell death should be avoided. I didn't. Similarly, there was the problem that the Aβ42 peptide did not change until after the disease had progressed. In addition, although cytokines can be measured with high sensitivity, they have the drawback of poor disease specificity. Furthermore, although the terminal Man-Tf has high specificity, it cannot be said that it is sufficient in terms of sensitivity.

WO2017 / 195778

The present invention develops a biomarker capable of specifically and accurately distinguishing the presence or absence of AD pathological condition from before the onset of the disease to the late stage of the onset of the disease and the risk of the onset of the disease, and a kit for detecting the biomarker, and a kit for detecting them. An object of the present invention is to provide a highly accurate method for differentiating the AD pathological condition used.

In order to solve the above problems, the present inventors have conducted intensive studies, and conducted in situ hybridization in the human cerebrum, mass analysis of mannose sugar chains bound to Tf in the brain, and immunohistochemical analysis of AD patients. In the hippocampus, we found that both terminal Man-Tf and p-tau, which are markers for AD diagnosis, are co-localized in neurons. This result suggests that terminal Man-Tf secretion and tau phosphorylation may be correlated. Therefore, as a result of investigating the relationship between sensitivity and specificity by combining the amounts of terminal Man-Tf protein and p-tau protein or tau protein in the cerebrospinal fluid collected from AD patients and MCI patients, the product of both was obtained, respectively. It was found that it is an excellent differential marker for AD pathology as compared with the case of using it alone. In addition, other known AD diagnostic markers are available for transferase glycoproteins (terminal GlcNAc-Tf proteins) having N-acetylglucosamine non-reducing terminal sugar chains and prostaglandin-H2 D-isomerase (PGDS). It was found that when combined, they can be excellent biomarkers for differentiating AD pathology compared to the case of each alone.

The present invention has been developed based on these new findings, and provides the following. (1) A combination marker for differentiating Alzheimer's disease pathological condition consisting of any of the following (a) to (c).
(A) Transtransferase glycoprotein (Man-Tf protein) containing at least one sugar chain having mannose at the non-reducing end, a fragment thereof containing the sugar chain having mannose, and a sugar chain having N-acetylglucosamine at the non-reducing end. Any one selected from the group consisting of a transferase (GlcNAc-Tf protein) containing only a fragment thereof or a fragment thereof containing the sugar chain having N-acetylglucosamine, and a prostaglandin D2 synthase (PGDS) or a fragment thereof. , And a combination of any one of the polypeptides selected from the group consisting of tau protein (tau protein), phosphorylated tau protein (p-tau protein) and fragments thereof.
(B) At least two or more selected from the group consisting of the Man-Tf protein or a fragment thereof containing a sugar chain having the mannose, the GlcNAc-Tf protein or a fragment thereof containing the sugar chain, and the PGDS or a fragment thereof. (C) The Man-Tf protein or a fragment thereof containing the sugar chain having the mannose, or the fragment thereof containing the GlcNAc-Tf protein or the sugar chain having the N-acetylglucosamine, and the AD index. In combination, the AD index is calculated from the formula (amyloid β40 peptide / amyloid β42 peptide) × p-tau protein.
(2) In the transtransferase (Tf) protein consisting of the amino acid sequence shown in SEQ ID NO: 1, a sugar chain having mannose at the non-reducing end of the Man-Tf protein is added to the asparagine residue at position 432, and the GlcNAc- The differential combination marker according to (1), wherein the sugar chain having N-acetylglucosamine at the non-reducing end of the Tf protein is added to the asparagine residues at positions 432 and 630.
(3) The differential combination marker according to (1) or (2), wherein the Alzheimer's disease condition comprises Alzheimer's disease (AD), mild cognitive impairment (MCI), and a healthy control (NC).
(4) The differential combination marker according to (3), which further differentiates the NC into an MCI transitional healthy control (pre-MCI) and an MCI non-transitional healthy control (non-pre-MCI).
(5) A peptide-binding molecule and a sugar chain bond that are a kit for differentiating Alzheimer's pathology and specifically bind to each of the combination markers for differentiating Alzheimer's pathology according to any one of (1) to (4). The polypeptide containing the molecule and specifically bound to the peptide bond molecule is at least one selected from the group consisting of Tf protein, PGDS, and tau protein, and the sugar chain binding molecule specifically binds to the polypeptide. The above-mentioned discrimination kit, wherein the sugar chain is a sugar chain having mannose at the non-reducing end and / or a sugar chain having N-acetylglucosamine at the non-reducing end.
(6) The differentiation kit according to (5), wherein the peptide bond molecule is an antibody or an active fragment thereof, or an aptamer.
(7) The discrimination kit according to (5) or (6), wherein the sugar chain-binding molecule is selected from the group consisting of lectins, antibodies or active fragments thereof, and aptamers.
(8) The method for differentiating Alzheimer's disease pathology according to any one of (1) to (4), which is present in a predetermined amount of body fluid obtained from a subject suspected of having dementia. The amount of each marker in the combination marker for use was measured using a peptide bond molecule that specifically binds to the polypeptide of the marker and a sugar chain binding molecule that specifically binds to the sugar chain of the marker, and the measured value thereof. The step of obtaining the product value obtained by multiplying the measured values of the respective markers, and the step of obtaining the AD, MCI, from the product value based on the preset AD cutoff value and MCI cutoff value. The discrimination method including a step of determining which of NC and NC can be applied.
(9) A subject who is determined to be NC by the method for differentiating Alzheimer's disease according to (8) based on a preset pre-MCI cutoff value, which is a method for differentiating a healthy control with MCI transition. The discrimination method including a step of determining whether the subject can correspond to pre-MCI or non-pre-MCI from the product value.
(10) A combination marker for differentiating Alzheimer's disease pathological condition, which comprises any of the following (a) to (c).
(A) Transtransferase glycoprotein (Man-Tf protein) containing at least one sugar chain having mannose at the non-reducing end, a fragment thereof containing the sugar chain having mannose, and a sugar chain having N-acetylglucosamine at the non-reducing end. Any one selected from the group consisting of a transferase (GlcNAc-Tf protein) containing only a fragment thereof or a fragment thereof containing the sugar chain having N-acetylglucosamine, and a prostaglandin D2 synthase (PGDS) or a fragment thereof. , And a combination of any one of the polypeptides selected from the group consisting of tau protein (tau protein), phosphorylated tau protein (p-tau protein) and fragments thereof.
(B) At least two or more selected from the group consisting of the Man-Tf protein or a fragment thereof containing a sugar chain having the mannose, the GlcNAc-Tf protein or a fragment thereof containing the sugar chain, and the PGDS or a fragment thereof. (C) The Man-Tf protein or a fragment thereof containing the sugar chain having the mannose, or the fragment thereof containing the GlcNAc-Tf protein or the sugar chain having the N-acetylglucosamine, and the AD index. It is a combination.
This specification includes the disclosure content of Japanese Patent Application No. 2020-163105, which is the basis of the priority of the present application.

According to the combination marker of the present invention, the AD pathological condition of the subject, that is, the presence or absence of AD or MCI and the possibility of being affected can be discriminated with high accuracy.

It is a conceptual diagram showing the structure of three kinds of Tf sugar chain isoforms of transferrin glycoprotein present in cerebrospinal fluid. A is a terminal Sia-Tf glycoprotein having two sialic acid non-reducing terminal sugar chains, B is a terminal GlcNAc-Tf glycoprotein having two GlcNAc non-reducing terminal sugar chains, and C is a Man non-reducing terminal sugar chain. And GlcNAc show a terminal Man-Tf glycoprotein having one non-reducing terminal sugar chain each. It is an immunohistochemical staining diagram showing the localization of Tf protein and phosphorylated tau (p-tau) protein in the hippocampus. A indicates the hippocampus of a healthy body, and B indicates the hippocampus of an AD patient. In the figure, merge is a diagram in which a staining diagram of Tf protein and p-tau protein is synthesized. It is a dot graph which shows the amount of Man-Tf protein in the cerebrospinal fluid in various dementia patients. In the figure, the black triangles indicate NC patients who have transitioned from NC to MCI by follow-up, and the black squares indicate MCI patients who have transitioned from MCI to AD by follow-up. It is a dot graph in each AD pathological condition when two markers are combined. A is a dot graph for comparison when only Man-Tf protein is used as a marker, B is a dot graph when a combination marker of Man-Tf protein and p-tau protein is used, and C is Man-Tf protein. The dot graph when the combination marker of tau protein is used is shown. The broken line in the figure shows the cutoff value of each group when iNPH is used as a disease control.

1. 1. Combination marker for Alzheimer's disease pathology differentiation (combination marker for AD pathology discrimination)
1-1. Overview A first aspect of the present invention is a combination marker for differentiating Alzheimer's disease (often referred to herein as "combination marker for AD pathology" or simply "combination marker"). The combination marker of the present invention comprises a combination of two or more kinds of proteins or peptide fragments thereof, and can be used in the AD pathological condition differentiation method of the third aspect described later for a subject suspected of having dementia.

1-2. Definition In the present specification, "Alzheimer's disease state" (often referred to as "AD condition" in the present specification) is a tauopathy whose pathogenic mechanism is abnormal accumulation of phosphorylated tau protein in nerve cells. In, refers to a group exhibiting symptoms of Alzheimer's disease or similar mild symptoms. Specifically, this includes Alzheimer's disease and mild cognitive impairment (MCI).

As mentioned above, "Alzheimer's disease (AD)" is an irreversible progressive central nervous system disease that presents with symptoms such as dementia, behavioral disorders, or personality changes.

"Mild cognitive impairment (MCI)" means that one of the cognitive functions (memory, decision, reasoning, execution, etc.) is impaired, and as a result, a slight decrease in cognitive function is observed. However, it is a condition that does not interfere with daily life. As used herein, MCI is included in central nervous system diseases. As mentioned above, some MCI patients are known to develop AD as their condition progresses.

In the present specification, "normal control (NC)" is a patient who is suspected of having dementia who has visited a memory outpatient clinic and is diagnosed as neither AD nor MCI according to dementia clinical diagnostic criteria such as dementia score. It means something. Subjects diagnosed with NC are referred to herein as "NC patients".

In the present specification, "patient with suspected dementia" refers to all patients who have undergone a forgetfulness outpatient clinic. Patients with suspected dementia include AD, MCI, progressive supranuclear palsy (abbreviated as "PSP" in this specification), and frontotemporal dementia; FTD ”), dementia with Lewy bodies (abbreviated as“ DLB ”in the present specification), Parkinson's disease (abbreviated as“ PD ”in the present specification) and NC. It falls under either category and is usually divided into either group according to the diagnosis of a doctor according to clinical diagnostic criteria. The diagnostic criteria used in this case are well known to those skilled in the art, including the above-mentioned Dementia Score (MMSE) (see, for example, Dementia Disease Clinical Practice Guidelines 2017, Igaku-Shoin).

In the present specification, "mild cognitive impairment transition type NC (pre-MCI)" refers to a pathological condition among NCs that is likely to eventually transition to MCI as the condition progresses without therapeutic intervention. Conventionally, NC has been diagnosed as normal for dementia because it does not correspond to any dementia in the clinical diagnostic criteria for dementia. However, this time, the combination marker for AD pathological condition identification of the present invention has revealed that there may be a population in which a part of NC may be transferred to MCI in the future. Therefore, in the present specification, NC is considered to include two groups of pre-MCI and non-pre-MCI.

"Non-pre-MCI" is almost synonymous with conventional NC, and the possibility of transition to MCI in the future is low, and it may be normal for dementia. High NC.

As used herein, the term "diagnosis" refers to the differentiation or possibility of morbidity of a disease, or the determination of the pathophysiology of a disease. Usually, the diagnostic act is the exclusive business of a doctor, a veterinarian, or a dentist, but the diagnosis in the present specification includes an auxiliary act of assisting the diagnosis by a doctor or the like without going through the act of the doctor or the like. ..

As used herein, the term "affected () differentiation" refers to determining whether or not a person has a specific disease. It also includes distinguishing from other diseases with similar lesions and conditions.

As used herein, the term "possibility of morbidity" refers to the probability of shifting from the current state to a specific disease in the future and suffering from that specific disease. As used herein, it means the probability of contracting AD when a specific disease is AD, and in particular, the probability of transition from MCI to AD.

As used herein, the term "subject" refers to an object of application in each aspect of the present invention. Specifically, it refers to a subject to be subjected to the differentiation method of this embodiment for the purpose of differentiating the morbidity of AD pathology. In principle, it is an individual animal, but it also includes its tissues and cells. Specific examples thereof include mammals, preferably humans, dogs, cats, horses and the like. It is preferably human. In the case of an individual, it does not matter whether it is alive or dead, but it is preferably a living body. The individual may be either an individual suffering from some kind of disease, such as a person undergoing a medical examination, an individual having a possibility of suffering from some kind of disease, or a healthy body. The “disease-potential individual” is, for example, an individual having a possibility of transition from MCI to AD in the future or a pre-MCI individual having a possibility of transition from NC to MCI in the future.

In the present specification, the "healthy body" means an individual in a healthy state. As used herein, the term "healthy state" means at least a state not suffering from AD pathology or the like, preferably a healthy state without any disease or disorder. In addition, the "disease control" described in Examples described later is an individual suffering from idiopathic normal pressure hydrocephalus (idiopathic Normal Pressure Hydrocephalus; often abbreviated as "iNPH" in the present specification). iNPH is clearly different from AD pathology. Therefore, the disease control corresponds to a healthy body herein. In the present specification, a healthy body is mainly used as a control body as a reference for comparison with a subject. Therefore, in the present specification, the subject and the healthy body are the same species. Further, it is preferable that the conditions such as subspecies (including race), gender, age (including monthly and weekly age), height, and weight are the same.

As used herein, the term "body fluid" refers to a liquid sample collected from a subject and a control body. Examples thereof include cerebrospinal fluid (cerebrospinal fluid), blood (including serum, plasma and interstitial fluid), urine, lymph, digestive fluid, ascites, pleural effusion, perineural fluid, and extracts of tissues or cells. .. It is preferably cerebrospinal fluid or blood, more preferably cerebrospinal fluid.

As used herein, the term "cerebrospinal fluid (CSF)" refers to a colorless and transparent extracellular fluid that exists only around the central nervous system (CNS: Central Nerve System) of the brain and spinal cord. It is separated from the blood by the hard membrane and by the blood-cerebrospinal and blood-cerebrospinal fluid barriers. Most of the cerebrospinal fluid exudes from the parenchyma of the brain and between brain cells and cerebrospinal fluid. Recent studies have shown that there are virtually no barriers (Wang C, et al., 2012, Cerebrospinal Fluid: Physiology, biomarker and methodology. In: V.S, Dolezal, T., editor. Cerebrospinal Fluid: Functions, Composition and Disorders. New York: Nova Science Publishers; pp.1-37.). There are many proteins derived from the central nervous system in this cerebrospinal fluid, and they are associated with central nervous system diseases. It is known that the expression increases or decreases. For example, in AD pathology, it is suggested that the amount of cerebrospinal fluid increases and the waste products of the brain including Aβ are eliminated (Lliff, et al). ., Sci. Transl. Med., 2010, 4).

As used herein, the term "glycoprotein" refers to a protein to which one or more sugar chains are added. In glycoproteins, sugar chains are added as one of post-translational modifications, and are said to be present in more than 50% of proteins in vivo. Sugar chains play an important role in imparting various functions to proteins, such as being involved in protein stabilization, protection, bioactivity, antigen-antibody reaction, viral infection, and pharmacokinetics.

In the present specification, the "peptide fragment (including the sugar chain)" is a peptide consisting of a part of the glycoprotein and containing an amino acid residue to which the target sugar chain is added. For example, a peptide consisting of a part of terminal Man-Tf and containing an asparagine residue at position 432 to which a mannose non-reducing terminal sugar chain is added can be mentioned. The number of amino acids in the peptide fragment is not particularly limited. Since it is a part of glycoprotein, the lower limit may be a length that can include the epitope, for example, 8 amino acids or more, preferably 10 or 15 amino acids or more, more preferably 20 or 30 amino acids or more, and the upper limit is glycoprotein. It suffices to have amino acids less than the total length of.

As used herein, the term "marker" refers to an index molecule for differentiating the onset or possibility of morbidity of a disease. As used herein, the marker corresponds to a biomarker targeting a molecule derived from a living body, particularly a polypeptide (including a glycoprotein and a fragment thereof). The marker is detected in the body tissue of the subject, but is preferably detected in body fluids herein.

As used herein, the term "combination marker" refers to a marker that can achieve a predetermined purpose by combining a plurality of the markers. The term "predetermined purpose" as used herein refers to the differentiation of Alzheimer's disease.

In the present specification, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measured and control values of the subject when statistically processed. In the present invention, the significant difference between the measured value of the terminal Man-Tf amount of the subject and the healthy body group when statistically processed is applicable. For example, when the risk factor (significance level) of the obtained value is small, specifically, when it is smaller than 5% (p <0.05), when it is smaller than 1% (p <0.01), and when it is smaller than 0.1% (p). <0.001) can be mentioned. The "p (value)" shown here indicates the probability that the test statistic will happen to be the value in the distribution based on the null hypothesis in the statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic will be that value, and the more likely it is that the null hypothesis will be rejected. As the test method for statistical processing, a known test method capable of determining the presence or absence of superiority may be appropriately used, and is not particularly limited. For example, Student's t-test method, covariate ANOVA, etc. can be used.

1-3. Composition The "Combination marker for differentiating Alzheimer's disease (combination marker for AD pathological condition)" of the present invention is composed of a combination of markers consisting of two or more types of protein or peptide fragments, and is intended for subjects suspected of having dementia. It is a biomarker capable of differentiating the susceptibility of AD or MCI in AD pathology.

1-3-1. Markers Constituting Combination Markers The combination markers of the present invention are composed of two or more markers. The combination markers of the present invention are a transferrin glycoprotein or a fragment thereof containing a predetermined sugar chain, a prostaglandin D2 synthase or a fragment thereof, a tau-related protein or a fragment thereof, and an AD index. Hereinafter, each marker will be described.

(1) Transferrin glycoprotein (Tf glycoprotein)
As used herein, "transferrin glycoprotein" (often referred to herein as "Tf glycoprotein") is a transferrin protein to which an N-linked sugar chain has been added.

The transferrin (Tf) protein (often referred to herein as the "Tf protein") is a carrier with a molecular weight of approximately 80 KDa that reversibly binds to two iron (Fe) ions and is responsible for its in vivo transport. It is a protein. Specific examples of the Tf protein include a human Tf protein composed of 698 amino acid residues and consisting of the amino acid sequence shown in SEQ ID NO: 1.

There are three types of isoforms in which the protein part is common to Tf glycoproteins and only the structure of the added N-linked sugar chain is different. Specifically, it is a terminal Man-Tf protein, a terminal GlcNAc-Tf protein, and a terminal Sia-Tf protein. Of these, the Tf glycoproteins that can constitute the combination marker of the present invention are the terminal Man-Tf protein and the terminal GlcNAc-Tf protein. Each Tf glycoprotein will be specifically described below.

(I) Terminal Man-Tf protein A "terminal Man-Tf protein" is an N-linked sugar chain having mannose (often referred to as "Man" in the present specification) at the non-reducing end, that is, the Man non-reducing end. A Tf glycoprotein containing at least one sugar chain, which is a constituent marker of the combination marker of the present invention. For example, as shown in C of FIG. 1, a Tf glycoprotein having a structure in which a Man non-reducing terminal sugar chain is added to one of the two sugar chains and a GlcNAc non-reducing terminal sugar chain is added to the other can be mentioned. .. More specifically, in the case of a human-terminal Man-Tf protein, in the amino acid sequence shown in SEQ ID NO: 1, the side chain of the asparagine (Asn: N) residue at position 432 (denoted by "N432") is not Man. A Tf glycoprotein to which a reduced terminal sugar chain is added and a GlcNAc non-reduced terminal sugar chain is added to the side chain of the asparagine residue at position 630 (denoted by "N630") corresponds to this.

The terminal Man-Tf protein is considered to be biosynthesized in the brain because it is not detected in the cerebrospinal fluid in anencephaly in which most of the cerebrum is congenitally deleted.

The amount of terminal Man-Tf increases in AD pathology. That is, the amount of terminal Man-Tf in body fluid, mainly in cerebrospinal fluid, is increased as compared with a healthy body, and therefore, terminal Man-Tf alone can be a marker for differentiating AD pathological condition reflecting AD pathological condition. On the other hand, iNPH patients cannot be used as a differential marker for iNPH because there is no significant difference in the amount of terminal Man-Tf in body fluids, mainly in cerebrospinal fluid, as compared with healthy bodies. In addition, although it is not easy to distinguish the symptoms of early AD from the initial symptoms of DLB or FTD from symptomatic differences, even with DLB and FTD, the terminal Man- in body fluids, mainly in cerebrospinal fluid, is compared with that of healthy bodies. There is no significant difference in the amount of Tf. Therefore, AD can be easily distinguished from DLB or FTD by measuring the amount of terminal Man-Tf in body fluid.

A peptide fragment of a terminal Man-Tf protein containing a Man non-reducing terminal sugar chain can also be a constituent marker of the combination marker of the present invention.

(Ii) Terminal GlcNAc-Tf protein "Terminal GlcNAc-Tf protein" is an N-linked sugar chain having N-acetylglucosamine (often referred to as "GlcNAc" in the present specification) at the non-reducing end, that is, GlcNAc. It is a Tf glycoprotein containing only a non-reducing terminal sugar chain, and is a constituent marker of the combination marker of the present invention. For example, as shown in B of FIG. 1, a Tf glycoprotein to which two GlcNAc non-reducing terminal sugar chains are added can be mentioned. More specifically, in the case of the human terminal GlcNAcn-Tf protein, the asparagine (Asn: N) residue at position 432 (denoted by "N432") and the asparagine residue at position 630 in the amino acid sequence shown in SEQ ID NO: 1 This is a Tf glycoprotein in which a GlcNAc non-reducing terminal sugar chain is added to the side chain of the group (denoted by "N630"). The terminal GlcNAc-Tf protein is produced in cells of the central nervous system such as the brain and / or spinal cord and is considered to be a brain-type glycoprotein found mainly in the cerebrospinal fluid. These brain-type glycoproteins are also known as biomarkers that reflect the production of cerebrospinal fluid (Murakami, et al., Proc. Jpn. Acad., Ser.B, 2019, 95, 198-210. ).

Generally, the terminal GlcNAc-Tf protein is known to be a marker for iNPH differentiation (Japanese Patent Laid-Open No. 2010-121980). In iNPH patients, the amount of terminal GlcNAc-Tf protein in body fluids, mainly in cerebrospinal fluid, was significantly reduced as compared with healthy bodies. On the other hand, there is no significant difference in the amount of terminal GlcNAc-Tf protein in body fluids, mainly in cerebrospinal fluid, compared with healthy subjects in AD patients and MCI patients. Therefore, the terminal GlcNAc-Tf protein alone cannot be a marker for differentiating AD pathology.

A peptide fragment of a terminal GlcNAc-Tf protein containing a GlcNAc non-reducing terminal sugar chain can also be a constituent marker of the combination marker of the present invention.

(Iii) Terminal Sia-Tf protein The "terminal Sia-Tf protein" is an N-linked glycoprotein having α2,6 sialic acid at the non-reducing end, that is, a Tf glycoprotein containing only the Sia non-reducing terminal sugar chain. .. Although it is an isoform of Tf glycoprotein, it is not a constituent marker of the combination marker of the present invention. For example, as shown in A of FIG. 1, a Tf glycoprotein to which two Sia non-reducing terminal sugar chains are added can be mentioned. Terminal Sia-Tf is present in both serum and cerebrospinal fluid, but is also referred to as "serum Tf (glycoprotein)" because it is present in a particularly large amount in serum. Terminal Sia-Tf is considered to be a Tf glycoprotein biosynthesized in the liver due to its similarity in sugar chain structure to other glycoproteins.

Like the terminal Man-Tf protein, the terminal GlcNAc-Tf protein is not detected in the cerebrospinal fluid in anencephaly, so it is considered to be biosynthesized in the brain.

(2) Prostaglandin D2 synthase (PGDS)
"Prostaglandin D2 synthase (often referred to herein as" PGDS ") is an enzyme that catalyzes the reaction of prostaglandin endoperoxides to prostaglandin D2 (PGD ₂ ) and is the present invention. There are constituent markers for the combination markers. Specific examples of the PGDS protein include a human PGDS protein composed of 190 amino acid residues and composed of the amino acid sequence shown in SEQ ID NO: 2. It is known that PGDS has a relatively high content in cerebrospinal fluid, but a concentration of about 1% thereof can also be detected in blood. PGDS is also known as a biomarker that reflects the production of cerebrospinal fluid (Murakami, et al., Proc. Jpn. Acad., Ser.B, 2019, 95, 198-210.).

(3) tau-related protein The term "tau-related protein" as used herein refers to tau protein and phosphorylated tau protein (often referred to as "p-tau protein" in the present specification). Tau protein is a microtubule-associated protein involved in axonal transport and is present in nerve cells and glial cells of the central and peripheral nervous systems. In AD, tau protein is excessively phosphorylated to p-tau protein. It is thought that this p-tau protein inhibits the stability of microtubules and causes intracellular cell death by forming intracellular aggregates. Since both tau protein and p-tau protein are intracellular proteins, their detection in body fluid usually means neuronal cell death. It is known that tau-related proteins can be markers for dementia such as PSP and FTD in addition to AD and MCI, and are also used as AD markers in clinical practice.

Constituent marker of the combination marker of the present invention As a specific example of a certain tau protein, there is a human tau protein composed of 441 amino acid residues and composed of the amino acid sequence shown in SEQ ID NO: 3. In the human tau protein, in addition to the amino acid sequence shown in SEQ ID NO: 3, there are 6 types of isoforms consisting of 352 to 412 amino acid residues which are splicing variants thereof. These isoforms can also be constituent markers of the combination markers of the present invention. Phosphorylation occurs at various locations in p-tau proteins, and usually in AD, excessive phosphorylation occurs, so that phosphorylation can occur at multiple amino acid residues. Specifically, for example, expressed by the position in the amino acid sequence shown in SEQ ID NO: 3, the threonine residue at position 181, the threonine residue at position 199, the threonine residue at position 202, the threonine residue at position 205 or the position 231 Phosphorylation at amino acid residues including threonine residues and the like. Detection of p-tau can be performed, for example, by phosphorylation site-specific detection, site-non-specific phosphorylation detection, or a combination thereof.

(4) AD index
The "AD index" is a value usually calculated by (Aβ40 / Aβ42) × p-tau, and is a marker that reflects both metabolic changes of Aβ peptide and p-tau protein in AD. Further, for the calculation of the AD index, for example, (Aβ42 / Aβ40) × p-tau may be used, but in the present specification, the value calculated by any of the formulas is included. The AD index is considered to have the highest AD diagnostic ability by itself among the AD markers currently used clinically.

1-3-2. Combination of constituent markers The following are examples of combinations of constituent markers in the combination marker for AD pathological condition identification of the present invention.

(1) Combination of Man-Tf / GlcNAc-Tf / PGDS and tau / p-tau As the first combination pattern, it has Man-Tf protein or a fragment thereof containing a sugar chain having Man, GlcNAc-Tf protein or GlcNAc. A constituent marker consisting of a fragment thereof containing a sugar chain and any one of the polypeptides selected from the group consisting of PGDS or a fragment thereof, and any of the constituent markers selected from the group consisting of tau protein, p-tau protein and fragments thereof. Examples include a combination with a constitutive marker consisting of one polypeptide.

Here, the "fragment containing a sugar chain having Man" refers to a peptide fragment containing an asparagine residue to which a Man non-reducing terminal sugar chain is added in the Man-Tf protein. For example, a Man-Tf protein fragment containing N432 and / or N630 to which a Man non-reducing terminal sugar chain has been added.

Further, "a fragment containing a sugar chain having GlcNAc" refers to a peptide fragment containing an asparagine residue to which a Man non-reducing terminal sugar chain is added in the GlcNAc-Tf protein. For example, a Man-Tf protein fragment containing N432 and N630 added with a Man non-reducing terminal sugar chain.

A more specific combination of constituent markers is a Man-Tf protein or a fragment thereof containing a sugar chain having Man and a tau protein or a fragment thereof, a Man-Tf protein or a fragment thereof containing a sugar chain having Man and a p-tau protein. Or a fragment containing a phosphorylation site thereof, a fragment thereof containing a sugar chain having GlcNAc-Tf protein or GlcNAc and a tau protein or a fragment thereof, a fragment thereof containing a sugar chain having GlcNAc-Tf protein or GlcNAc and a p-tau protein or A fragment containing the phosphorylation site, and a PGDS protein or a fragment thereof and a tau protein or a fragment thereof, a PGDS protein or a fragment thereof and a p-tau protein or a fragment containing the phosphorylation site thereof.

(2) Two or more combinations in Man-Tf / GlcNAc-Tf / PGDS As the second combination pattern, a fragment containing a Man-Tf protein or a sugar chain having Man, a GlcNAc-Tf protein or a sugar chain having GlcNAc is used. Included is a combination of at least two proteins selected from a constitutive marker consisting of the fragment comprising and any one polypeptide selected from the group consisting of PGDS or fragments thereof.

A more specific combination of constituent markers is a combination of two constituent markers, a fragment thereof containing a Man-Tf protein or a sugar chain having Man, and a fragment thereof containing a GlcNAc-Tf protein or a sugar chain having GlcNAc, Man-Tf. A combination of a fragment thereof containing a sugar chain having a protein or Man and two constituent markers of PGDS or a fragment thereof, a combination of a fragment thereof containing a sugar chain having a GlcNAc-Tf protein or GlcNAc and a combination of two constituent markers of PGDS or a fragment thereof. , And a fragment thereof containing a Man-Tf protein or a sugar chain having Man, a fragment thereof containing a GlcNAc-Tf protein or a sugar chain having GlcNAc, and a combination of three constituent markers of PGDS or a fragment thereof.

Any of the above combination markers can distinguish AD and MCI from NC as AD pathology. It is also possible to distinguish between pre-MCI and non-pre-MCI in NC.

(3) Combination of Man-Tf / GlcNAc-Tf and AD index The third combination pattern includes a Man-Tf protein or a fragment thereof containing a sugar chain having Man, and a sugar chain containing a GlcNAc-Tf protein or GlcNAc. The combination of the fragment and the AD index can be mentioned.

2. 2. Alzheimer's disease pathological differentiation kit (AD pathological condition discrimination kit)
2-1. Overview A second aspect of the present invention is an Alzheimer's disease pathological differentiation kit. The kit of the present invention can detect the marker for AD pathological condition according to the first aspect that can be contained in the sample, thereby distinguishing the morbidity or possibility of morbidity of AD pathological condition of the subject.

2-2. Structure The AD diagnostic kit of the present invention contains a peptide bond molecule and a sugar chain bond molecule that specifically bind to each of the combination markers for AD pathological condition discrimination according to the first aspect as essential constituents. Hereinafter, each will be described in detail.

(1) Peptide bond molecule As used herein, the term "peptide bond molecule" refers to a molecule that specifically binds to a polypeptide or a peptide fragment thereof in a constituent marker of a combination marker for AD pathological condition differentiation. Examples of the constituent marker polypeptide, that is, the target polypeptide, include Tf protein, PGDS, and tau protein.

The AD diagnostic kit of the present invention contains one or more peptide bond molecules that bind to these polypeptides. The peptide bond molecule may be any of a peptide, nucleic acid, small molecule compound, or a combination thereof.

(I) Peptide bond molecule composed of a peptide Examples of the peptide bond molecule composed of a peptide include an antibody or an active fragment thereof.
The antibody may be either a polyclonal antibody, a monoclonal antibody or a recombinant antibody. Monoclonal or recombinant antibodies are preferred to allow for more specific detection. The globulin type of the antibody is not particularly limited and may be any of IgG, IgM, IgA, IgE, IgD and IgY, but IgG and IgM are preferable. Further, the species from which the antibody of this embodiment is derived is not particularly limited. It can be of any animal origin, including mammals and birds. Examples include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans.

As used herein, the term "recombinant antibody" refers to, for example, a chimeric antibody, a humanized antibody, and a synthetic antibody.

A "chimeric antibody" is an antibody in which the constant region of a light chain and a heavy chain (C region: Constant region) is replaced with the C region of a light chain and a heavy chain of another antibody. For example, in a mouse anti-human monoclonal antibody, an antibody in which the C region of the light chain and the heavy chain is replaced with the C region of an appropriate human antibody is applicable. That is, in this case, the variable region (V region: Variable region) including the CDR is derived from the mouse antibody, and the C region is derived from the human antibody.

A "humanized antibody" is also referred to as a reshaped human antibody, and is a mosaic antibody in which the CDR in an antibody derived from a non-human animal to a target antigen is replaced with the CDR of a human antibody. For example, a recombinant antibody gene was prepared by substituting a DNA sequence encoding each CDR region (CDR1 to CDR3) of a mouse anti-human Man-Tf antibody with a DNA sequence encoding each corresponding CDR derived from an appropriate human antibody. , Antibodies obtained by expressing it.

"Synthetic antibody" refers to an antibody synthesized by using a chemical method or a recombinant DNA method. For example, a monomeric polypeptide molecule in which one or more VLs and one or more VHs of a specific antibody are artificially linked via a linker peptide having an appropriate length and sequence, or a multimeric polypeptide thereof. Applicable. Specific examples of such polypeptides include single-chain Fv (scFv: single chain Fragment of variable region) (Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL) and diabody. ), Triabody, tetrabody and the like. In immunoglobulin molecules, VL and VH are usually located on separate polypeptide chains (light chain and heavy chain). Single-stranded Fv is a synthetic antibody fragment having a structure in which the V regions on these two polypeptide chains are linked by a flexible linker of sufficient length and contained in one polypeptide chain. Within a single-stranded Fv, both V regions can self-assemble with each other to form one functional antigen-binding site. Single-stranded Fv can be obtained by incorporating the recombinant DNA encoding it into the phage genome using a known technique and expressing it. Diabody is a molecule with a structure based on a single-stranded Fv dimer structure (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448). The triabodies and tetrabodies have trimer and tetramer structures based on a single-stranded Fv structure, similar to diabodies. They are trivalent and tetravalent antibody fragments, respectively, and may be multispecific antibodies.

The antibody preferably has a high affinity with a target polypeptide having a dissociation constant of 10 ^-8 M or less, preferably 10 ^-9 M or less, more preferably 10 ^-10 M or less. The dissociation constant can be measured using a technique known in the art. For example, it may be measured using the speed evaluation kit software by the BIAcore system (GE Healthcare).

The polyclonal antibody of this embodiment used in this step is known from immune animals in the art after immunizing a suitable animal with a polypeptide serving as an antigen, that is, Tf protein, PGDS, and tau protein, or a peptide fragment thereof. It can be recovered by the method. In addition, a monoclonal antibody can also be obtained by a known method which is a conventional technique in the art. For example, after immunizing a mouse with the antigen, antibody-producing cells are collected from the immune mouse. Hybridomas may be identified that fuse the antibody-producing cells to a myeloma cell line, thereby producing hybridoma cells and producing monoclonal antibodies that bind to the target polypeptide.

The "antibody fragment" is a peptide fragment having the antigen-binding activity of the above-mentioned antibody, and examples thereof include Fab, F (ab') 2, Fv and the like.

The antibody or antibody fragment thereof used in this step may be modified. The term "modification" as used herein includes a label required for antibody detection or a functional modification required for antigen-specific binding activation. Labeling includes, for example, the aforementioned fluorescent substances, fluorescent proteins (eg, PE, APC, GFP), enzymes (eg, horseradish peroxidase, alkaline phosphatase, glucose oxidase), or labeling with biotin or (streptavidin) avidin. .. In addition, modifications include glycosylation of antibodies performed to adjust the affinity for the target polypeptide. Specifically, for example, in the framework region (FR: Framework region) of an antibody, a substitution is introduced into an amino acid residue constituting a glycosylation site to remove the glycosylation site, whereby glycosylation at that site is removed. There are modifications that cause loss of glycosylation.

The "active fragment thereof" refers to a partial fragment of an antibody that retains antigen-binding property and immune response activity.

Specific antibodies in the AD diagnostic kit of the present invention include, for example, anti-Tf antibody, anti-PGDS antibody, anti-tau antibody, anti-p-tau antibody, anti-Aβ40 antibody, and anti-Aβ42 antibody.

(Ii) Peptide bond molecule composed of nucleic acid Examples of the peptide bond molecule composed of nucleic acid include nucleic acid aptamers.
A "nucleic acid aptamer" is an aptamer composed of nucleic acids, which is strong with a target substance due to the secondary structure of a single-stranded nucleic acid molecule via hydrogen bonds and the three-dimensional structure formed based on the tertiary structure. And a ligand molecule that has the ability to specifically bind.

The nucleic acid aptamer used in the present specification can be produced by a method known in the art. For example, an in vitro sorting method using a SELEX (systematic evolution of ligands by exponential enrichment) method can be mentioned. The SELEX method is, for example, in the case of separating RNA aptamers, "selecting an RNA molecule bound to a target molecule from an RNA pool composed of a large number of RNA molecules having a random sequence region and primer binding regions at both ends thereof. After amplifying the recovered RNA molecule by RT-PCR reaction, transcription is performed using the obtained cDNA molecule as a template to obtain an amplification product of the selected RNA molecule, which is used as the RNA pool for the next round. This is a method of selecting an RNA molecule having stronger binding force to a target molecule by repeating a series of cycles of "" for several to several tens of rounds. On the other hand, when the DNA aptamer is separated by the SELEX method, the basic procedure is the same, but it is not necessary to go through the RT-PCR reaction when amplifying the recovered DNA molecule. The base sequence lengths of the random sequence region and the primer binding region are not particularly limited. Generally, the random sequence region is preferably in the range of 20 to 80 bases, and the primer binding region is preferably in the range of 15 to 40 bases, respectively. In order to increase the specificity to the target molecule, a molecule similar to the target molecule and an RNA pool or an RNA pool are mixed in advance, and a pool consisting of an RNA molecule or a DNA molecule that does not bind to a molecule similar to the target molecule is formed. It may be used. The SELEX method is a known method, and the specific method may be, for example, according to Pan et al. (Proc. Natl. Acad. Sci. 1995, U.S.A. 92: 11509-11513). In the present invention, the target molecule can be used as a sugar chain mannose, and the nucleic acid molecule finally obtained by carrying out the above method can be used as a nucleic acid aptamer for mannose.

As nucleic acid aptamers, RNA aptamers and DNA aptamers are generally known, but the nucleic acids constituting the nucleic acid aptamers in the present specification are not particularly limited. For example, it includes a DNA aptamer, an RNA aptamer, an aptamer composed of a combination of DNA and RNA, and the like. Generally, RNA aptamers are frequently used, but DNA aptamers are superior in terms of stability, production cost in chemical synthesis, and the number of steps in aptamer production.

Nucleic acid aptamers used in this step include fluorescent substances (eg, FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluoresamine and so on, as long as they do not inhibit the ability to bind to the target molecule. It can also be labeled with its derivatives, such as rhodamine and its derivatives), radioactive isotopes (eg, 32P, 33P, 35S), or labeling substances such as biotin or (streptavidin) avidin.

Specific examples of the nucleic acid aptamer in the AD diagnostic kit of the present invention include Tf protein-binding RNA aptamer, PGD-binding RNA aptamer, tau protein-binding RNA aptamer, and p-tau protein-binding RNA aptamer.

The peptide bond molecule in the AD diagnostic kit of the present invention may be immobilized on a carrier or labeled with a fluorescent dye, a luminescent substance, or the like, if necessary.

(2) Glycoprotein-binding molecule As used herein, the “sugar chain-binding molecule” refers to a molecule that specifically binds to the sugar chain of a glycoprotein in a constituent marker of a combination marker for AD pathological condition differentiation. Examples of the constituent marker polypeptide, that is, the target glycoprotein, include Man-Tf protein and GlcNAc-Tf protein, which are Tf glycoproteins. The AD diagnostic kit of the present invention contains one or more sugar chain-binding molecules that bind to the sugar chains of these Tf glycoproteins. The sugar chain binding molecule may be any of a peptide, a nucleic acid, a small molecule compound, or a combination thereof.

(I) Sugar chain-binding molecule composed of a peptide Examples of the sugar chain-binding molecule composed of a peptide include a lectin, an antibody, or an active fragment thereof.

(Lectin)
"Lectin" refers to a protein or glycoprotein that binds to a sugar chain other than an immune response. The lectins that can be included in the Alzheimer's disease pathological identification kit of the present invention include sugar chains containing Man or GlcNAc, preferably sugar chains containing terminal Man or terminal GlcNA, and more preferably Man non-reducing terminal sugar chains or GlcNA non-reducing terminals. Examples include lectins that bind to sugar chains.

When the non-reducing end of the sugar chain is Man, a Man non-reducing end sugar chain-binding lectin can be mentioned. Specific examples of Man non-reducing terminal sugar chain-binding lectins include 45 types of Man-binding lectins described in http://jcggdb.jp/rcmg/glycodb/LectinSearch including UDA lectins and BC2L-A lectins. Can be mentioned. In addition, Hori et al. Separated a large number of non-reducing terminal Man-binding lectins obtained from algae, and their use is also conceivable (Bioscience and Industry, 71, 129-133, 2013).

When the non-reducing end of the sugar chain is GlcNAc, GlcNA non-reducing end sugar chain-binding lectin can be mentioned. Specific examples of the GlcNA non-reducing terminal sugar chain-binding lectin include, for example, the agglutinin GSL-II lectin derived from the mushroom family Griffonia simplicifolia, the agglutinin ABA lectin derived from Agaricus bisporus, and the agglutinin derived from the child body of Psathyrella velutina. The agglutinin PVL lectin is known.

By the way, in lectins, there may be a difference in apparent sugar chain binding specificity between the case where the target molecule is a glycoprotein and the case where the target molecule is only a sugar chain. For example, UDA lectins derived from urtica (Urtica dioica) are classified as GlcNAc-binding lectins when the target molecule is a sugar chain, but when the target molecule is a glycoprotein, they do not bind to the terminal GlcNAc-Tf protein. It becomes specifically bound to the Man non-reduced end of the terminal Man-Tf protein. Therefore, it is preferable to examine the binding of the lectin using not only the sugar chain but also the terminal Man-Tf protein or the terminal GlcNAc-Tf protein which is a target molecule. That is, the lectin constituting the kit of the present invention is Man of the terminal Man-Tf protein or GlcNac of the terminal GlcNAc-Tf protein, preferably terminal Man of the terminal Man-Tf protein or terminal GlcNAc of the terminal GlcNAc-Tf protein, more preferably. Is a lectin that binds to the non-reducing terminal Man of the terminal Man-Tf protein or the non-reducing terminal GlcNAc of the terminal GlcNAc-Tf protein. As the lectin, a commercially available lectin may be used. For example, biotinylated UDA lectin (Cat No. BA-8005-1; EY) and BC2L-A lectin derived from bacteria (Burkholderia cenocepacia lectin-A; Wako Pure Chemical Industries, Ltd.) can be used.

(Antibody or active fragment thereof)
The composition of the antibody or its active fragment is specifically described in "(1) Peptide bond molecule, (i) Peptide bond molecule composed of peptide" described above, and the same applies to this invention. Then, the explanation is omitted. Specific examples of the antibody that binds to the sugar chain include a sugar chain containing Man, preferably a sugar chain containing a terminal Man, and more preferably an antibody that recognizes and binds to a sugar chain containing a non-reducing terminal Man. Examples thereof include an antibody that recognizes and binds to a sugar chain containing GlcNAc, preferably a sugar chain containing a terminal GlcNAc, and more preferably a sugar chain containing a non-reducing terminal GlcNAc.

(Ii) Sugar chain-binding molecule composed of nucleic acid Examples of the sugar chain-binding molecule composed of nucleic acid include nucleic acid aptamers.
The composition of the nucleic acid aptamer and the method for producing the same are specifically described in the above-mentioned "(1) Peptide bond molecule, (ii) Peptide bond molecule composed of nucleic acid", and the same applies to the present invention. Then, the explanation is omitted.
The nucleic acid-binding molecule in the AD diagnostic kit of the present invention may be immobilized on a carrier or labeled with a fluorescent dye, a luminescent substance, or the like, if necessary.

Most of the AD therapeutic agents (especially monoclonal antibodies against Aβ) that are being developed these days suppress the decline in cognition. Therefore, these therapeutic agents may be most effective when administered to patients in the early stage of onset to suppress the progression of symptoms at an early stage. For this reason, it is most desirable to administer to pre-MCI patients with dementia. However, there has been no established predictive differential marker before the onset of MCI. The combination marker of the present invention is not only an excellent marker for differentiating AD pathology, but also enables differentiation of pre-MCI patients, and is expected to contribute to early diagnosis and treatment of AD in the future.

3. 3. Alzheimer's disease pathological differentiation method (AD pathological condition discrimination method)
3-1. Overview A third aspect of the present invention is a method for differentiating AD pathological conditions. In the method for differentiating AD morbidity of the present invention, the amount of the combination marker for differentiating AD pathology of the first aspect present in the body fluid derived from a subject suspected of having dementia is measured, and the subject is AD or MCI based on the measured value. , And NC can be determined.

3-2. Method The AD pathological condition discrimination method of the present invention includes a measurement step, a product value calculation step, and a determination step as essential steps. Hereinafter, each step will be specifically described.

(1) Measurement step In the “measurement step”, the amount of each marker in the combination marker for AD pathological condition discrimination according to the first aspect present in a predetermined amount of body fluid obtained from a subject suspected of having dementia is measured. , Is the process of obtaining the measured value.

The body fluid is preferably cerebrospinal fluid or blood. The method for collecting cerebrospinal fluid and blood may be any known method and is not particularly limited. For example, if it is cerebrospinal fluid, it may be collected by lumbar puncture. Lumbar puncture is relatively less invasive because pain can be reduced to less than blood sampling by using a commercially available local anesthetic in advance, and side effects can be reduced by using a traumatic needle. This is a suitable method for collecting cerebrospinal fluid. If it is blood, it may be collected according to a known blood collection method. As a general rule, the body fluids of the subject and the control body should be the same kind of body fluids as if one is cerebrospinal fluid and the other is cerebrospinal fluid.

"Predetermined amount" means an amount predetermined by capacity or weight. Although the predetermined amount is not particularly limited, it is necessary that the marker for AD pathological condition discrimination according to the first aspect contained at least in the body fluid of the subject, preferably in the cerebrospinal fluid, is a measurable amount. For example, the amount of cerebrospinal fluid may be 5 μL to 1 mL, or the amount of cerebrospinal fluid protein may be 5 μg to 200 μg.

In the present specification, the "measured value" is a value indicating the amount of each marker in the combination marker for AD pathological condition discrimination measured in this step. The measured value may be an absolute value such as volume or weight, or may be a relative value such as concentration, ionic strength, absorbance or fluorescence intensity.

The amount of the marker is determined by using a peptide-binding molecule that specifically binds to the polypeptide of each constituent marker of the combination marker for AD pathological condition differentiation and a sugar chain-binding molecule that specifically binds to the sugar chain of the constituent marker. Just measure. Since the configurations of the peptide bond molecule and the sugar chain bond molecule are described in detail in the second aspect, specific description thereof will be omitted here.

The measuring method may be any known protein or glycoprotein quantification method using a peptide bond molecule and a sugar chain binding molecule, and is not particularly limited. For example, an immunological detection method using an antibody, a lectin detection method using a lectin, a mass spectrometry method or a combination method thereof can be mentioned.

Immunological detection methods include, for example, enzyme immunoassay (including ELISA and EIA methods), fluorescent immunoassay, radioimmunoassay (RIA), luminescence immunoassay, and surface plasmon resonance (SPR). , Crystal transducer microbalance (QCM) method, immunoturbidimetric method, latex agglutination immunoassay, latex turbidimetric method, hemagglutination reaction, particle agglutination reaction method, gold colloid method, capillary electrophoresis method, western blot method or immunity Histochemical method (immunostaining method) can be mentioned.

Examples of the lectin detection method include a lectin blotting method.
High-speed liquid chromatograph mass spectrometry (LC-MS), high-speed liquid chromatograph tandem mass spectrometry (LC-MS / MS), gas chromatograph mass spectrometry (GC-MS), gas chromatograph tandem mass spectrometry include mass spectrometry. Analytical methods (GC-MS / MS), capillary electromass spectrometry (CE-MS) and ICP mass spectrometry (ICP-MS) can be mentioned.

As the combination method, for example, a sandwich ELISA method using a lectin and an antibody and an automated method using a similar principle, preferably a high-throughput lectin inhibition-automatic lattice aggregation method can be used. When the constituent marker of the combination marker for AD pathological condition identification is glycoprotein, the combination method of detecting each of the sugar chain and the protein as a target is preferable because the glycoprotein can be quantified with high accuracy. As a specific example of the combination method, when the terminal Man-Tf protein is measured as one of the constituent markers in the combination marker for AD pathological condition differentiation, a body fluid (for example, spinal fluid) is passed through a carrier such as a plate on which an anti-Tf antibody is adsorbed. A method of adsorbing the terminal Man-Tf protein and then detecting the terminal Man-Tf protein using the UDA lectin, which is a Man-binding molecule, as a probe can be mentioned. On the contrary, there is also a method of adsorbing the terminal Man-Tf protein through a body fluid (for example, cerebrospinal fluid) on a carrier such as a plate on which UDA lectin is adsorbed, and then detecting the terminal Man-Tf protein with an anti-Tf antibody.

All of the above analysis methods are techniques known in the art, and may be performed according to those methods. For example, Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Christopher J., et al. : 1103-1169 ； Iijima Y. et al., 2008 ,. The Plant Journal, 54, 949-962 ； Hirai M. et al., 2004, Proc Natl Acad Sci USA, 101 (27) 10205-10210 ； Sato S, et It can be performed according to the method described in al., 2004 ,, The Plant Journal, 40 (1) 151-163; Shimizu M. et al., 2005, Proteomics, 5, 3919-3931. In addition, protein quantification kits are commercially available from each manufacturer, and they can also be used.

(2) Product value calculation step The “product value calculation step” is a step of obtaining a product value obtained by multiplying the measured values of the respective markers obtained in the measurement step.

(3) Judgment step The "judgment step" determines whether the subject can correspond to AD, MCI, or NC from the product value based on the preset AD cutoff value and MCI cutoff value. It is a process.

In the present specification, the "cutoff value" refers to a value that can determine the presence or absence of the risk of illness or the pathological condition based on the value. Preferably, the cutoff value indicates a sufficiently high value for both sensitivity and specificity. Generally, it is derived from the ROC curves drawn based on a direct comparison between the control group and the disease group to be compared using a known method, but is not limited to this. It does not have to be a direct comparison between the control group and the disease group to be compared, and the cutoff value may be set without using the ROC curve.

The "ROC curve (Receiver Operating Characteristic curve)" means that the vertical axis is the true positive rate (TPF: True Position Fraction), that is, the sensitivity and the horizontal axis is the false positive rate (FPF: False Position Fraction). That is, it is set to (1-specificity), and it is created by changing and plotting which value of the test result is judged to have a finding, that is, the cutoff point as a parameter. Here, the specificity is the rate at which a negative person is accurately judged to be negative.

The "AD cutoff value" is a cutoff that serves as a reference for determining whether or not a subject subject to the discrimination method of the present invention is likely to be affected by AD, based on the product value obtained in the product value calculation step. The value. If the product value is higher than the AD cutoff value, the subject is determined to be more likely to have AD. If the product value is lower than the AD cutoff value, the subject is determined to be less likely to have AD and more likely to be either MCI or NC.

The specific value of the AD cutoff value differs depending on the combination of constituent markers in the combination marker for AD discrimination. The AD cutoff values for each combination are shown in Tables 1 to 4. For example, when the combination marker for AD discrimination is a combination of Man-Tf protein and p-tau protein, the AD cutoff value is 97.8 from Table 1.

The "MCI cutoff value" is a cutoff that serves as a reference for determining whether or not a subject subject to the discrimination method of the present invention is likely to be affected by MCI, based on the product value obtained in the product value calculation step. The value. If the product value is higher than the MCI cutoff value, the subject is determined to be more likely to have MCI. If the product value is lower than the AD cutoff value, it is determined that the subject is likely to be NC.

The specific value of the MCI cutoff value differs depending on the combination of constituent markers in the combination marker for AD pathological condition discrimination. The MCI cutoff values for each combination are shown in Tables 1-4. For example, when the combination marker for AD pathological condition differentiation is a combination of Man-Tf protein and p-tau protein, the MCI cutoff value is 110 from Table 1.

The AD pathological condition differentiation method of this embodiment can be carried out in place of or in combination with a known AD pathological condition discrimination method. Known methods for differentiating AD pathology include, for example, measurement of dementia score, amyloid PET (positron emission tomography) using [ ¹¹ C] Pittsburgh compound (PiB), and fluoro-2-. PET using deoxy-D-glucose (FDG) and the like can be mentioned.

4. Differentiation method for MCI transition type healthy control (pre-MCI discrimination method)
4-1. Overview A fourth aspect of the present invention is a method for differentiating a healthy control with MCI transfer (pre-MCI differentiating method). The pre-MCI discrimination method of the present invention can determine whether the subject is NC or non-MCI or non-pre-MCI.

4-2. Method The pre-MCI discrimination method of the present invention includes a measurement step, a product value calculation step, a determination step, and a re-judgment step. Of these, the measurement step, the product value calculation step, and the determination step are the same as the AD pathological condition discrimination method of the third aspect. That is, the pre-MCI differentiation method of the present invention is a differentiation method that follows the AD pathological condition differentiation method of the third aspect. Hereinafter, the re-determination step, which is a feature of the present invention, will be described.

(4) Re-judgment step The "re-judgment step" is a subject who is determined to correspond to NC in the determination step in the AD pathological condition discrimination method of the third aspect based on a preset pre-MCI cutoff value. This is a step of determining whether the subject can correspond to pre-MCI or non-pre-MCI from the product value obtained in the product value calculation step.

The "pre-MCI cut-off value" is a cut that serves as a criterion for determining whether an NC has a high possibility of MCI transition or an NC that does not, when the subject is determined to be NC by the AD pathological condition discrimination method of the third aspect. Off value.

Subjects determined to be AD or MCI in the determination step in the AD pathological condition differentiation method of the third aspect are not subject to the pre-MCI differentiation method of the present invention, and only subjects determined to be NC are targeted. If the product value obtained in the product value calculation process is higher than the pre-MCI cutoff value, it is determined that the subject is pre-MCI and is currently NC but is likely to move to MCI in the future. On the other hand, if the product value is lower than the pre-MCI cutoff value, the subject is determined to be non-pre-MCI, that is, true NC, which is unlikely to move to MCI.
The pre-MCI differentiation method of this embodiment can be used in combination with a known AD pathological condition differentiation method or the like.

<Example 1: Localization of Tf protein and p-tau protein in the hippocampus>
(Purpose)
To verify the localization of the Man-Tf protein, known as an AD marker, in the hippocampus.
(Method)
Since the hippocampus, which controls short-term memory, is the site where nerve cell death and tissue atrophy progress at the earliest stage, AD patients and healthy hippocampus for control are double-stained with anti-Tf protein antibody and anti-p-tau antibody. Was done. 5 micron sections were made from formalin-fixed hippocampus and immunostained on glass slides. Anti-Tf protein antibody (A0061, Dako Ltd.) and anti-p-tau protein antibody (AT8, Cosmo Bio Co., Ltd.) are used as the primary antibody, and Alexa Fluor 488 or 594 labeled antibody (Thermo Fisher Scientific) is used as the secondary antibody. ) Was used. It is clear from the results of mass spectrometry that the Man-Tf protein occupies 85% or more of the sugar chain isoform in the Tf protein of the cerebral cortex.

(result)
The results are shown in FIG. The Tf protein was detected in both healthy subjects (FIG. 2A) and AD patients (FIG. 2B), but was detected slightly more strongly in AD patients than in healthy subjects. On the other hand, p-tau protein was not detected in healthy subjects, but was detected in AD patients.
Interestingly, some of the Tf protein-positive cells were also p-tau protein-positive cells, as indicated by the arrows (Fig. 2B). These results suggest that Tf protein secretion increases with the progression of AD pathology, and that Tf protein and p-tau protein are co-localized.

It is known that when cells are exposed to stress and the amount of mishold protein increases, high mannose sugar chains become tags and recalibration of the higher-order structure of the protein occurs (Helenius A. and Aebi M., Ann. Rev. Biochem. ., 2004, 73: 1019-1049). From this, the increase in Man-Tf protein may be due to the stress response.

<Example 2: Measurement of Man-Tf protein in cerebrospinal fluid>
(Purpose)
The amount of Man-Tf protein in the cerebrospinal fluid was measured, and the effect of Man-Tf protein as a marker on various dementias was verified.

(Method)
1. 1. Diagnosis of each disease For 316 patients who visited the outpatient department for dementia, diagnosis was made by the existing diagnostic method, and 5 mL of cerebrospinal fluid was collected from each patient by lumbar puncture.
1-1. Diagnosis of AD pathology and other neurodegenerative diseases Patients suspected of having outpatient AD were classified into MCI and AD according to the following AD diagnostic criteria, and the group of patients who did not belong to either was designated as NC. At the same time, tauopathy other than AD, which accumulates tau protein, which is a dementia with neurodegenerative disease, and synucleinopathy, which accumulates α-synuclein, were also classified according to the following diagnostic criteria. In the following examples, tauopathy and synucleopathy are often referred to as "other neurodegenerative diseases".
Idiopathic normal pressure hydrocephalus (iNPH) showed dementia and ventricular enlargement like AD, but did not show neurodegenerative disease, so it was used as a disease control.

(1) Diagnosis of AD According to the clinical diagnostic criteria by the National Institute on Aging-Alzheimer's association workgroup (NIA-AA).
(2) Diagnosis of MCI The clinical diagnostic criteria of MCI against the background of AD recommended by the NIA-AA Diagnostic Guideline Development Workgroup were followed.
(3) Diagnosis of tauopathy Specific diseases of tauopathy include progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD). For these diagnoses, the dementia disease clinical practice guideline 2017 (Igaku-Shoin electronic version ISBN 978-4-260-62858-7) was followed.
(4) Diagnosis of synucleinopathy Specific diseases of synucleinopathy include Lewy body dementia (DLB) and Parkinson's disease (PD). For DLB, we followed the Dementia Disease Practice Guidelines 2017 (supra). For PD, the Parkinson's disease clinical practice guideline 2018 (Igaku-Shoin) was followed.
(5) Disease-controlled diagnosis The diagnosis of iNPH was in accordance with the 3rd edition of the Idiopathic Normal Pressure Hydrocephalus Clinical Practice Guideline (Medical Review).

2. 2. Measurement of Man-Tf protein in cerebrospinal fluid In the measurement of Man-Tf protein, rBC2L-A lectin was used as a sugar chain binding molecule. The capture reaction was carried out by immobilizing an anti-human Tf antibody on a plate. Specifically, anti-human Tf antibody (A0061, Dako Ltd) was diluted to 1 μg / mL with 100 mM carbonate buffer (pH 9.5), 100 μL was added to a microtiter plate and incubated overnight at 4 ° C. After washing once with TBS, TBS containing 10% N101 (Fuji Film Wako Pure Chemical Industries, Ltd.) was added as a blocking agent and incubated at room temperature for 1 hour to prepare an antibody-immobilized plate.

Cerebrospinal fluid (5-10 μL) of the sample is placed at 55 ° C. for 60 minutes before in PBST (phosphate-buffered saline / 0.05% Tween-20) containing 0.6% 2-mercaptoethanol and 0.003% SDS at a final concentration. Processing was performed. The blocked antibody-immobilized plate was washed once with TBST, then the pretreated sample was diluted with TBST (TBST-CaCl ₂ ) containing 10 mM CaCl ₂ and incubated overnight at 4 ° C. After washing 3 times with TBST-CaCl ₂ , 100 μL of TBST-CaCl ₂ containing biotinylated rBC2L-A (50 ng / mL) was added, and the mixture was incubated at room temperature for 2 hours. rBC2L-A was previously biotinylated using Ez-link NHS-biotin (# 21336, Pierce) according to the attached protocol. TBST-CaCl ₂ was added and washed twice, 100 μL of TBST-CaCl ₂ containing HRP-labeled streptavidin (50 ng / mL) was added, and the mixture was incubated at room temperature for 2 hours. After washing twice with TBST-CaCl ₂ , 100 μL of TMB Micro well Peroxidase substrate system (# 50-76-11, Kirkegaard & Perry Laboratories, Inc.) reagent was added and a color reaction was carried out. The reaction was stopped with 100 μL of 1 M phosphoric acid, and the absorbance was measured at 450 nm using a Varioskan LUX multimode microplate reader (Thermo Fischer Scientific). At this time, as a standard for quantification, human serum Tf (cat No. T1147, Sigma-Aldrich) having a sialic acid α2,6 galactose β1,4GlcNAcβ1, (3/6) -mannose residue at the end of the sugar chain was used as a sialidase. A Tf sugar chain isomer having a mannose residue at the end of the sugar chain, which was prepared by sequential digestion with galactosidase and hexosaminidase, was used.
Statistical analysis was performed by SPSS (version 26). After one factor ANOVA analysis, multiple comparisons were performed by Turkey-Kramer comparison.

(result)
The results are shown in FIG. As a result of multiple tests, NC (3.56 + 1.46 μg / mL), MCI (3.72 + 1.54 μg / mL) and AD (2.99 + 1.01 μg / mL) for the disease control iNPH (2.12 + 0.71 μg / mL) The Man-Tf protein was significantly increased in. On the other hand, no increase was observed in other neurodegenerative diseases (PSP + FTD and DLB + PD).
In addition, according to the follow-up survey, 5 of the 23 cases in the NC group were transferred to MCI (black triangle in the figure), and 2 of the 42 cases in the MCI group were transferred to AD (black square in the figure).
From the above results, it was suggested that the measured value of Man-Tf protein increased specifically in AD pathology, especially in NC and MCI. Furthermore, it was suggested that MCI patients who could transition to AD from the distribution of black squares could be predicted from the measured values of Man-Tf protein. On the other hand, as is clear from the distribution of the black triangle, it is suggested that it is difficult to predict NC patients (pre-MCI patients) who can migrate to MCI by the measured values of Man-Tf protein alone.

<Example 3: Improvement of discrimination accuracy by combining AD markers>
(Purpose)
We will verify whether the accuracy of differentiating AD pathology can be improved by combining two types of AD markers.

(Method)
From the results of Example 1, it was suggested that the p-tau protein and the Tf protein co-localize in the same nerve cell. Therefore, these two types of AD markers were combined and the amount of each was measured. In the onset of AD, it is assumed that amyloid plaques (deposition of Aβ protein) first occur 20 years or more before the onset of AD, and then p-tau protein deposits occur about 10 years after that. Therefore, we also verified the combination of AD index and Tf protein calculated from Aβ peptide and p-tau protein. The following ELISA kit was used for the measurement of tau protein, p-tau protein, Aβ40 peptide, and Aβ42 peptide.
-Measurement of tau protein (including phosphorylation at the threonine residue at position 181):
FinoScholar ・ hTAU 10-992 FinoScholar ・ pTAU (Nipro)
According to the manufacturer's recommendation, 400 pg / mL or more was positive.
・ Measurement of p-tau protein:
FinoScholar / hTAU 10-994 FinoScholar / pTAU (Nipro)
According to the manufacturer's recommendation, 50 pg / mL or less was negative.
-Measurement of Aβ40 peptide:
Human / rat βamyloid (40) ELISA Kit, Wako II (cat No. 294-64701, Wako Pure Chemical Industries, Ltd.)
-Measurement of Aβ42 peptide:
Human / rat βamyloid (42) ELISA Kit, Wako, High Sensitive (cat No. 292-64501, Wako Pure Chemical Industries, Ltd.)
The AD index was calculated by (Aβ40 / Aβ42) × p-tau based on the measured amount of p-tau protein, Aβ40 peptide, and Aβ40 peptide.

The cutoff value of the combination marker was obtained based on the product value obtained by multiplying the measured values of each marker. The cutoff values for AD and MCI were set from the ROC curve using the Yoden method. On the other hand, for the pre-MCI cutoff value in NC, the cutoff value obtained based on the comparison between "patients with suspected AD (NC + MC + AD)" and "disease control (iNPH)" was used in a pseudo manner. The sensitivity and specificity were calculated from the cutoff value, and the AUC (area under the curve) representing the reliability was calculated using the receiver operating characteristic (ROC) curve.

(result)
The results are shown in Table 1, Table 2, and FIG. 4 (sensitivity and specificity are shown in bold at 70% or higher, and AUC is shown at 0.75 or higher in bold).

(1) Accuracy of discrimination between MCI and AD by combination marker As shown in Fig. 4A and Table 1, when only Man-Tf protein is used as a marker, the MCI group is more sensitive to the MCI cutoff value of 2.67 μg / mL. The specificity was 81.0% and the specificity was 82.7%. The AUC for evaluating the reliability of the ROC curve was 0.846. Similarly, in the AD group, the specificity was 82.7% but the sensitivity was 67.2% due to the AD cutoff value of 2.62 μg / mL. In AUC, it was 0.776 in MCI. From this result, it was suggested that the accuracy of differentiating AD pathology is not sufficient when only Man-Tf protein is used as a marker.

Even when only p-tau protein or only tau protein is used as a marker, as shown in Table 1, the sensitivity (p-tau: 87.5%, tau: 84.8%) and specificity (p-tau: 96.2) and specificity (p-tau: 96.2) in the AD group. %, Tau: 96.2%) and AUC exceeded 0.9, but in the MCI group, the specificity (p-tau: 96.2%, tau: 96.2%) was high, but the sensitivity was considerably low. (P-tau: 59.4%, tau: 44.4%). Therefore, it was suggested that the p-tau protein or tau protein alone does not have sufficient discrimination accuracy.

Therefore, the sensitivity, specificity, and AUC were calculated from the combination of Man-Tf protein and p-tau protein. As shown in FIG. 4B, the MCI group has an MCI cutoff value (110a.u .: dashed line) based on the product value (p-tau × Man-Tf) of the measured values of Man-Tf protein and p-tau protein. As a result of the verification, as shown in Table 1, the sensitivity was 83.9%, the specificity was 90.4%, and the AUC was 0.919. Generally, an AUC above 0.9 is considered to be extremely accurate. In addition, as a result of verification using the AD cutoff value (98a.u .: broken line) based on the product value in the AD group, the sensitivity was as high as 93.9%, the specificity was as high as 88.5%, and the AUC was also extremely high as 0.957. Therefore, it was shown that the combination of Man-Tf protein and p-tau protein provides extremely high accuracy for the differentiation of MCI and AD.

Similarly, the sensitivity, specificity, and AUC were calculated for the combination of Man-Tf protein and tau protein. As shown in FIG. 4C, the MCI group was verified with the MCI cutoff value (559 a.u .: broken line), and as shown in Table 1, both the sensitivity was 88.9% and the specificity was 88.5%, and the AUC was 0.907. there were. In the AD group, as a result of verification with the AD cutoff value (615 a.u .: broken line), as shown in Table 1, the sensitivity is 100% and the specificity is 90.4%, both of which are extremely high, and the AUC is also extremely high at 0.962. showed that. Therefore, it was shown that the combination of Man-Tf protein and tau protein also provides extremely high accuracy for the differentiation of MCI and AD.

(2) Application of combination marker to NC group As described in Example 2, it has been clarified by follow-up survey that a part of NC group is transferred to MCI. However, it was difficult to predict and distinguish between NC patients (pre-MCI patients) who could migrate to MCI and NC patients (non-pre-MCI) who did not migrate to MCI by the measured values of Man-Tf protein alone. Therefore, the cutoff value set based on the product value was applied to each marker measured value in the combination marker to the NC group.

As a result of verification with pre-MCI cutoff values (99.4 and 559, respectively) based on the respective product values when using the combination marker of p-tau × Man-Tf or tau × Man-Tf, as shown in Table 1. In addition, the sensitivity (86.6 and 91.9, respectively) and specificity (88.5 and 80.4, respectively) exceeded 80%, and the AUC (0.931 and 0.918, respectively) exceeded 0.9 in each combination, which was excellent before the onset of MCI. It was shown that it can be a predictive discrimination marker. In addition, the product value of Man-Tf protein or GlcNAc-Tf protein and AD index also provides high accuracy for the differentiation of MCI and AD, and can be an excellent predictive differentiation marker before the onset of MCI. It has been shown.

On the other hand, when this cutoff value is applied to other neurodegenerative disease groups (PSP + FTD + DLB + PD), the ratio above the cutoff value is 28% for p-tau x Man-Tf and tau x Man-Tf. In the case of 37%, both were very low, indicating that the increase in the value of these products was AD pathologically specific.

From the above, it was shown that the combination marker of Man-Tf protein, p-tau protein and tau protein is an extremely excellent marker in sensitivity and specificity in the differentiation of AD pathology and pre-MCI. The

<Example 4: Verification of AD discrimination accuracy using other combination markers>
(Purpose)
The accuracy of differentiating AD pathological conditions by a combination marker other than Example 3 will be verified.

(Method)
Using a part of the cerebrospinal fluid sample of Example 3, the amount of GlcNAc-Tf protein and PGDS was measured by the following method.
The measurement of GlcNAc-Tf protein basically followed the method for detecting Man-Tf protein. First, a plate on which rPVL (recombinant Psathyrella velutina lectin, Medical and Biological Laboratories Co., Ltd.) was immobilized was prepared, rPVL (1 μg / 0.1 mL) was added to the plate, and the mixture was incubated overnight at 4 ° C. Next, 50 mM TBS containing 10% Block Ace (KAC Co., Ltd.) was added as a blocking agent, and blocking was performed at 4 ° C. for 4 hours.

Next, the blocked biotin-immobilized plate was washed once with TBST, the pretreated sample was added, and the mixture was incubated overnight at 4 ° C. After washing twice with TBST, 100 μL of anti-Tf antibody (A0061, Dako Ltd.) solution (0.5 μg / mL) was added, and the mixture was incubated at room temperature for 2 hours. After washing twice with TBST, 100 μL of a 20,000-fold diluted Western wasabi peroxidase-labeled anti-rabbit IgG antibody (Promega, W4011) solution (0.1 μg / mL) was added, and the mixture was incubated at room temperature for 2 hours. After washing twice with TBST, 100 μL of a 2-fold diluted TMB Microwell Peroxidase substrate system (# 50-76-11, Kirkegaard & Perry Laboratories, Inc., MD) reagent was added to perform a color reaction. The reaction was stopped by adding 100 μL of 1M phosphoric acid, and the absorbance was measured at 450 nm with a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, K.K., Tokyo). At this time, as a standard for quantification, human serum Tf (cat No. T1147, Sigma-Aldrich) having a sialic acid α2,6 galactose β1,4GlcNAcβ1, (3/6) -mannose residue at the end of the sugar chain was used as a sialidase and A Tf sugar chain isomer having a β1,4GlcNAcβ1, (3/6) -mannose residue at the end of the sugar chain prepared by sequential digestion with galactosidase was used.

A commercially available ELISA kit (Human prostaglandin D synthase (Lipocalin-type) ELISA (Cat. No .: RD191113100R, BioVendor)) was used to measure the amount of PGDS.
The cutoff value was set, the sensitivity, specificity, and AUC were calculated according to Example 4.

(result)
The results are shown in Tables 3 and 4.

GlcNAc-Tf protein and PGDS concentrations were measured in the cerebrospinal fluid and compared with the iNPH group. As shown in Table 3, in the case of GlcNAc-Tf protein alone, there was a significant increase in the NC, MCI and AD groups, but the sensitivity and specificity were not high. In the case of PGDS alone, a significant increase was observed only in the AD group, but the sensitivity and specificity were not high, and the AUC was less than 80%, so it was hard to say that it was a good marker.

On the other hand, in the case of the cutoff value based on the product value of GlcNAc-Tf protein and p-tau protein or tau protein, and the product value of PGDS and p-tau protein or tau protein, it is carried out as shown in Table 3. Similar to Example 3, the sensitivity and specificity of all AD pathologies were high, and the AUC was significantly higher than 0.8. Therefore, it was shown that the combination marker of the present invention can discriminate AD pathological conditions with high accuracy.

Further, as in Example 3, when the cutoff value based on the product value of the combination markers of the present embodiment is applied to other neurodegenerative disease groups (PSP + FTD + DLB + PD), the ratio of the cutoff value or more is any. It was as low as 26% to 37%, indicating that the increase in these product values is AD pathologically specific.

Furthermore, in order to investigate the usefulness of the combination marker in samples other than cerebrospinal fluid, the serum PGDS concentration was measured in iNPH individuals and MCI individuals by the same method as in this example. As previously known, PGDS alone did not show a clear difference in the amount between iNPH and MCI. Therefore, the product of the measured value of p-tau used in the above analysis of the same individual and the PGDS concentration in serum was calculated and used as a pseudo combination marker. Then, when this product value was used, the marker value was higher in MCI than in iNPH, and a significant tendency was observed between the two. This suggests that the combination marker is useful even when the measurement results from samples other than cerebrospinal fluid are used.

<Example 5: Verification of AD discrimination accuracy by different AD indexes>
(Purpose)
We will verify the accuracy of differentiating the AD pathology of the combination marker when the formula (Aβ42 / Aβ40) × p-tau is used for the AD index.
(Method)
The procedure was the same as in Example 3 except for the calculation of the AD index.
As the AD index, the value calculated by the formula (Aβ42 / Aβ40) × p-tau was used in this example.

(result)
The results are shown in Table 5 (sensitivity and specificity are shown in bold for 70% or higher, and AUC is shown in bold for 0.75 or higher).

When only the above AD index was used as a marker, the sensitivity was 83.3%, the specificity was 79.3%, and the AUC was 0.826 in the MCI group. However, in the AD group, the sensitivity was as high as 100%, but the specificity was as low as 65.5%. Therefore, it was suggested that the AD index alone does not have sufficient discrimination accuracy.

Therefore, the sensitivity, specificity, and AUC were calculated from the combination of the Man-Tf protein and the above AD index. When the product of the Man-Tf protein and the measured value of the above AD index was used as a marker, the sensitivity was 100% and the specificity was 72.4% in the AD group, which could not be sufficiently differentiated by themselves, and the discrimination accuracy was improved. However, AUC also showed 0.872. Furthermore, even in the MCI group, which originally had high discrimination accuracy, the sensitivity was 88.9% and the AUC was 0.895, showing even higher values.

When the product of the measured values of the Man-Tf protein and the above AD index was applied to the NC group, it also showed excellent discrimination accuracy compared to the case of each alone, and became an excellent predictive discrimination marker before the onset of MCI. Shown to get.
In addition, when the above AD index was used, the same results as when (Aβ40 / Aβ42) × p-tau were used as the AD index were obtained. Therefore, the combination marker of the present invention is a calculation formula of the AD index. Regardless, it was shown that it is a highly accurate differential marker for MCI and AD, and can be an excellent predictive differential marker before the onset of MCI.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (10)

1. A combination marker for differentiating Alzheimer's disease pathological condition consisting of any of the following (a) to (c).
   (A) Transtransferase glycoprotein (Man-Tf protein) containing at least one sugar chain having mannose at the non-reducing end, a fragment thereof containing the sugar chain having mannose, and a sugar chain having N-acetylglucosamine at the non-reducing end. Any one selected from the group consisting of a transferase (GlcNAc-Tf protein) containing only a fragment thereof or a fragment thereof containing the sugar chain having N-acetylglucosamine, and a prostaglandin D2 synthase (PGDS) or a fragment thereof. , And a combination of any one of the polypeptides selected from the group consisting of tau protein (tau protein), phosphorylated tau protein (p-tau protein) and fragments thereof.
   (B) At least two or more selected from the group consisting of the Man-Tf protein or a fragment thereof containing a sugar chain having the mannose, the GlcNAc-Tf protein or a fragment thereof containing the sugar chain, and the PGDS or a fragment thereof. (C) The Man-Tf protein or a fragment thereof containing the sugar chain having the mannose, or the fragment thereof containing the GlcNAc-Tf protein or the sugar chain having the N-acetylglucosamine, and the AD index. It is a combination of.
2. AD index is the differential combination marker according to claim 1, which is calculated from the formula of (amyloid β40 peptide / amyloid β42 peptide) × p-tau protein.
3. In the transferase (Tf) protein consisting of the amino acid sequence shown in SEQ ID NO: 1, a sugar chain having mannose at the non-reducing end of the Man-Tf protein is added to the asparagine residue at position 432, and the GlcNAc-Tf protein The differential combination marker according to claim 1 or 2, wherein the sugar chain having N-acetylglucosamine at the non-reducing end is added to the asparagine residues at positions 432 and 630.
4. The differential combination marker according to any one of claims 1 to 3, wherein the Alzheimer's disease condition comprises Alzheimer's disease (AD), mild cognitive impairment (MCI), and a healthy control (NC).
5. The differential combination marker according to claim 4, further differentiating the NC into an MCI transitional healthy control (pre-MCI) and an MCI non-transitional healthy control (non-pre-MCI).
6. A kit for differentiating Alzheimer's disease
   A peptide-binding molecule and a sugar chain-binding molecule that specifically bind to each of the combination markers for differentiating Alzheimer's disease according to any one of claims 1 to 5 are included.
   The polypeptide to which the peptide bond molecule specifically binds is at least one selected from the group consisting of Tf protein, PGDS, and tau protein.
   The above-mentioned discrimination kit, wherein the sugar chain to which the sugar chain-binding molecule specifically binds is a sugar chain having mannose at the non-reducing end and / or a sugar chain having N-acetylglucosamine at the non-reducing end.
7. The discrimination kit according to claim 6, wherein the peptide bond molecule is an antibody, an active fragment thereof, or an aptamer.
8. The discrimination kit according to claim 6 or 7, wherein the sugar chain-binding molecule is selected from the group consisting of lectins, antibodies or active fragments thereof, and aptamers.
9. It is a method of differentiating the pathological condition of Alzheimer's disease.
   The amount of each marker in the combination marker for differentiating Alzheimer's disease pathological condition according to any one of claims 1 to 5, which is present in a predetermined amount of body fluid obtained from a subject suspected of having dementia, is the marker. The step of measuring using a peptide bond molecule that specifically binds to the polypeptide of the above and a sugar chain binding molecule that specifically binds to the sugar chain of the marker and obtaining the measured value, and multiplying the measured value of each of the above markers. A step of obtaining the product value obtained from the product, and a step of determining whether the subject can correspond to AD, MCI, or NC from the product value based on the preset AD cutoff value and MCI cutoff value. The above-mentioned discrimination method including.
10. It is a method for differentiating MCI transition type healthy controls.
    Based on the preset pre-MCI cutoff value, the subject is pre-MCI and non-pre- The discrimination method including a step of determining which of the MCIs can be applied.

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