Methods for diagnosing and / or treating alzheimer's disease

Abstract

Methods for diagnosing and / or treating Alzheimer's disease are provided. Also provided are biomarkers and combinations of biomarkers that can diagnose the presence and progression of Alzheimer's disease and the treatment thereof.

Description

Technical Field

[0001] The present disclosure generally relates to methods for diagnosing and treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with unknown etiology and limited diagnostic markers. Clinical pathological studies have shown that AD pathology (particularly the accumulation of amyloid plaques) begins 10-20 years before the onset of cognitive symptoms. However, a definitive diagnosis of AD is still only available through neuropathological evaluation at autopsy. Data suggest that the pathogenesis of AD is early and insidious, and its clinical manifestations become apparent only after massive neuronal cell death and synaptic loss have occurred. However, current treatment modalities are limited by imperfect diagnostic parameters and the complete inability to identify early pathogenic disease processes.

[0003] Early diagnosis is an important step in treating Alzheimer's disease because it allows subjects to receive medications that can slow the progression of the disease. However, accurate diagnosis can be difficult, especially for patients with mild or early-stage Alzheimer's disease. A definitive diagnosis can be made by examining the pathology of brain tissue, but this is only possible after death.

[0004] Therefore, diagnostic methods are needed to identify and treat Alzheimer's disease. Summary of the invention

[0005] The present disclosure provides a method for diagnosing and / or treating Alzheimer's disease or alleviating its symptoms in a human subject in need, the method comprising determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase. The level of the one or more biomarkers can be determined and compared with the level of the one or more biomarkers in a normal individual (e.g., a reference sample from an individual without Alzheimer's disease, including, for example, an individual with normal levels of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase). The CDR of a normal individual can be 0, or the MMSE score of a normal individual can be about 27-30. Optionally, if the level of the one or more biomarkers of the subject is increased compared to the level of the one or more biomarkers in a normal individual, the method can further include the step of administering Alzheimer's treatment.

[0006] In an embodiment, the present disclosure provides a method of diagnosing and / or treating Alzheimer's disease in a subject in need thereof, the method comprising determining the presence of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein detection of the one or more biomarkers indicates that the subject has Alzheimer's disease, and administering an Alzheimer's disease treatment if the one or more biomarkers are detected in the sample (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected).

[0007] In some embodiments, the present disclosure provides methods of diagnosing and / or treating Alzheimer's disease in a subject in need thereof, the method comprising selecting a subject with Alzheimer's disease in need of such treatment by determining the presence of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein detection of the one or more biomarkers indicates that the subject has Alzheimer's disease, and administering an Alzheimer's disease treatment if the one or more biomarkers are detected (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected).

[0008] In certain embodiments, the present disclosure provides a method of identifying a human subject with Alzheimer's disease in need of treatment, the method comprising identifying a human subject with Alzheimer's disease in need of such treatment by determining the levels of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein detection of the one or more biomarkers indicates that the subject has Alzheimer's disease, and if the one or more biomarkers are detected (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected), administering an Alzheimer's disease treatment.

[0009] In a further embodiment, the present disclosure provides a method for selecting a subject for treatment of Alzheimer's disease or alleviation of symptoms thereof, the method comprising determining the level of one or more biomarkers (selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, selecting the subject for treatment of Alzheimer's disease or alleviation of symptoms thereof if the one or more biomarkers are detected (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected), and optionally administering an Alzheimer's disease treatment to the selected subject.

[0010] In yet further embodiments, the present disclosure provides a method of diagnosing and / or treating a subject at risk for developing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein detection of the one or more biomarkers indicates that the subject is at risk for developing Alzheimer's disease, and optionally, administering an Alzheimer's disease treatment if the one or more biomarkers are detected (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected).

[0011] In a further embodiment, the present disclosure provides a method for diagnosing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from a subject, and if the one or more biomarkers are detected (e.g., both phosphorylated FYN 416 and phosphorylated p38 MAP kinase are detected), diagnosing Alzheimer's disease in the subject.

[0012] In yet a further embodiment, the present disclosure provides a method for determining whether a subject is at risk for developing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein detection of the one or more biomarkers (e.g., detection of both phosphorylated FYN 416 and phosphorylated p38 MAP kinase) indicates that the subject is at risk for developing Alzheimer's disease.

[0013] In some embodiments, the present disclosure provides a method of treating Alzheimer's disease or alleviating a symptom thereof in a human subject in need thereof, the method comprising selecting a human subject with Alzheimer's disease in need of such treatment by determining an increased level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein the increase is measured as compared to normal levels of the one or more biomarkers (e.g., phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase) in a normal individual (e.g., an individual without Alzheimer's disease, including, for example, an individual with a CDR of 0 or an MMSE score of about 27-30), and administering an Alzheimer's disease treatment to the subject.

[0014] In a further embodiment, the present disclosure provides a method of treating Alzheimer's disease or alleviating a symptom thereof in a human subject in need thereof, the method comprising identifying a human subject with Alzheimer's disease in need of such treatment by determining an increased level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, wherein the increase in the level of the one or more biomarkers is measured as compared to the level of the one or more biomarkers (e.g., phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase) in a normal individual (e.g., an individual without Alzheimer's disease, including, for example, an individual with a CDR of 0 or an MMSE score of about 27-30), and administering an Alzheimer's disease treatment.

[0015] In yet a further embodiment, the present disclosure provides a method for selecting a subject for treatment of Alzheimer's disease or alleviation of symptoms thereof, the method comprising determining the level of one or more biomarkers (selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject; comparing the level of the one or more biomarkers with normal phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase levels for an individual (including, for example, an individual with a CDR of 0 or an MMSE score of about 27-30), selecting a subject for treatment of Alzheimer's disease or alleviation of symptoms thereof if the one or more biomarkers are increased relative to an individual with normal levels of the one or more biomarkers, and optionally administering an Alzheimer's disease treatment to the selected subject.

[0016] In a further embodiment, the present disclosure provides a method of treating a subject at risk for developing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (e.g., phosphorylated FYN416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, comparing the level of the one or more biomarkers to the level of the one or more biomarkers in individuals not at risk for developing Alzheimer's disease, and administering an Alzheimer's disease treatment if the one or more biomarkers are increased in the subject relative to the individuals not at risk for developing Alzheimer's disease.

[0017] In a further embodiment, the present disclosure provides a method for measuring the effectiveness of a treatment for Alzheimer's disease, the method comprising identifying a subject having a CDR score greater than 0 or an MMSE score less than 27, determining pre-treatment levels of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, administering an Alzheimer's disease treatment, and determining post-treatment levels of the one or more biomarkers in a sample collected from the subject, wherein a decrease in the post-treatment level of the biomarker indicates that the treatment is effective in treating Alzheimer's disease.

[0018] In a further embodiment, the present disclosure provides a method for diagnosing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from a subject, comparing the level of the one or more biomarkers to normal phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase levels in an individual (including, for example, an individual with a CDR of 0 or an MMSE score of about 27-30), and diagnosing Alzheimer's disease in the subject if the one or more biomarkers are detected.

[0019] In yet a further embodiment, the present disclosure provides a method for determining whether a subject is at risk for developing Alzheimer's disease, the method comprising determining the level of one or more biomarkers (e.g., phosphorylated FYN 416 and phosphorylated p38 MAP kinase) in a sample collected from the subject, comparing the level of the one or more biomarkers with the level of the one or more biomarkers in individuals not at risk for developing Alzheimer's disease, and identifying the subject as being at risk for developing Alzheimer's disease if the one or more biomarkers are increased in the subject relative to individuals not at risk for developing Alzheimer's disease.

[0020] In some embodiments, the present disclosure provides a method for diagnosing and / or treating Alzheimer's disease in a subject, wherein the method further comprises determining one or more biomarkers selected from phosphorylated Tau protein and / or B-amyloid protein. In certain embodiments, phosphorylated Tau protein includes phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231 and / or phosphorylated Tau 235. In some embodiments, beta-amyloid protein includes beta-amyloid protein 40 and beta-amyloid protein 42. In certain embodiments, relative to an individual (including, for example, an individual subject with a CDR of 0 or an MMSE score of about 27-30), the ratio of beta-amyloid protein 42 to beta-amyloid protein 40 of the subject increases.

[0021] In some embodiments, the level of one or more biomarkers is determined by immunology, including, for example, using antibodies specific for the one or more biomarkers. In certain embodiments, the level of one or more biomarkers is determined by immunohistochemistry, Western blot, ELISA, immunoprecipitation and / or flow cytometry analysis.

[0022] In a further embodiment, the level of one or more biomarkers is determined by quantitative immuno-PCR. Therefore, in an embodiment, the present disclosure provides a method for determining the level of one or more biomarkers of the present disclosure, which is performed by: contacting a sample with a ligand that is specific to the one or more biomarkers, wherein the ligand is connected to an oligonucleotide, and the oligonucleotide comprises a primer sequence unique to the biomarker, to allow the ligand to bind to the biomarker in the sample to form a complex, the complex comprising the ligand, the biomarker and the oligonucleotide, separating the complex, and amplifying the oligonucleotide. In some embodiments, the ligand and the oligonucleotide are non-covalently connected by a joint. In a further embodiment, the ligand is an antibody, the oligonucleotide is biotinylated, and the joint comprises a streptavidin-protein A chimera. In a further embodiment, the oligonucleotide is amplified by RT-PCR. In some embodiments, the oligonucleotide is amplified by digital droplet PCR. In certain embodiments, the oligonucleotide further comprises a unique barcode identifying the oligonucleotide, and wherein the oligonucleotide is sequenced to identify the connected ligand.

[0023] In some embodiments, the level of each of phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid is detected in a sample.

[0024] In some embodiments, the Alzheimer's disease treatment is one or more of the following: (galantamine), (rivastigmine), (donepezil), (memantine), or a pharmaceutically acceptable salt or ester thereof. In a specific embodiment, the Alzheimer's disease treatment is Aducanumab (Aducanumab, Aduhelm TM ).

[0025] In some embodiments, the present disclosure provides a method for diagnosing and treating Alzheimer's disease or alleviating symptoms thereof in a human subject, the method comprising determining an increased level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid, wherein the increased level is measured compared to the level of the biomarker in an individual without Alzheimer's disease, and if the level of the one or more biomarkers in the sample is increased, optionally administering an Alzheimer's disease treatment. DETAILED DESCRIPTION

[0026] The present disclosure provides methods for diagnosing and / or treating Alzheimer's disease. Surprisingly, the disclosed biomarkers and combinations of biomarkers can diagnose Alzheimer's disease and progression. Therefore, in embodiments, the present disclosure provides a group of proteins whose phosphorylation and / or isoform profiles are diagnostic for Alzheimer's disease and can therefore be used to identify subjects for Alzheimer's disease treatment, as well as to measure the effectiveness of Alzheimer's disease treatment.

[0027] definition

[0028] Before describing the present invention in detail, some definitions of terms used herein are provided. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0029] When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purpose of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising".

[0030] The use of "consisting of" or "consisting essentially of" language in the claims may further limit the specific embodiments disclosed herein. When used in a claim, whether as filed or added by amendment, the transition term "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the present disclosure as claimed are inherently or expressly described and enabled herein.

[0031] Where numerical values ​​are indicated in the context of the present disclosure, the skilled person will understand that the technical effect of the feature in question is ensured within an accuracy interval that generally encompasses a deviation of ±10%, and preferably ±5%, of the given numerical value. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0032] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (such as molecular weight and median particle size), reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0033] Unless otherwise specified herein or clearly contradictory to the context, the terms "one", "a", "said" and similar references used in the context of describing the present disclosure (especially in the context of the appended claims) will be interpreted as covering the singular and the plural. The numerical ranges listed herein are intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise specified herein, each individual value is incorporated into the specification as if the value was listed separately in this article. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure, rather than to limit the scope of the present disclosure otherwise claimed. Any language in the specification should not be interpreted as indicating any unclaimed elements essential to the practice of the present disclosure.

[0034] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member can be mentioned and claimed individually or with other members of the group or other elements found herein in any combination. For convenience and / or patentability reasons, it can be expected that one or more members of the group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, the specification is deemed to contain the group of modification, thereby satisfying the written description of all Markush groups used in the appended claims.

[0035] Certain embodiments of the present disclosure are described herein, including the best mode known to the inventor for implementing the present disclosure. Of course, after reading the above description, variations of the embodiments described will become apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will appropriately adopt such variations, and the inventor hopes to put the present disclosure into practice in a manner different from that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents to the subject matter listed in the attached claims where permitted by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, the present disclosure covers any combination of the above-mentioned elements in all possible variations thereof.

[0036] It will be understood that the embodiments of the present disclosure disclosed herein are illustrations of the principles of the present disclosure. Other modifications that may be adopted are within the scope of the present disclosure. Therefore, by way of example and not limitation, the optional configurations of the present disclosure may be used according to the teachings herein. Therefore, the present disclosure is not limited to exactly as shown and described.

[0037] Although the present disclosure is described and illustrated herein by reference to various specific materials, procedures and embodiments, it should be understood that the present disclosure is not limited to the specific combination of materials and procedures selected for this purpose. As will be appreciated by those skilled in the art, many variations of such details may be implied. This is intended to treat the specification and embodiments as merely exemplary, and the true scope and spirit of the present disclosure are indicated by the appended claims. All references, patents and patent applications mentioned in this application are incorporated herein by reference in their entirety.

[0038] Further definitions of the terms will be given below in the context of using these terms. The following terms or definitions are provided only to aid in understanding the present invention. The scope of these definitions should not be interpreted as less than that understood by a person of ordinary skill in the art.

[0039] As used herein, phosphorylated FYN 416 protein refers to a form of FYN src tyrosine kinase protein that is phosphorylated on tyrosine 416. FYN proteins (e.g., Genbank Accession Nos. NP_001357458.1, NM_002037.5, and isoforms thereof) are non-receptor tyrosine protein kinase members of the src tyrosine kinase family and play a role in many biological processes, including regulation of cell growth and survival, cell adhesion, integrin-mediated signaling, cytoskeletal remodeling, cell motility, immune responses, and axon guidance.

[0040] As used herein, phosphorylated p38 MAPK refers to a form of p38 mitogen-activated protein kinase that is phosphorylated on one or both of the dual activation residues in the classic threonine-glycine-tyrosine activation motif. p38 MAPK proteins (e.g., Genbank accession numbers NP_620583.1, NM_139014.3, and isoforms thereof) function as signal transduction nexus in many biological processes, such as the production of proinflammatory cytokines, control of connective tissue remodeling under pathological conditions, regulation of oxidation, and control of cell adhesion.

[0041] As used herein, phosphorylated Tau refers to the phosphorylated form of tau protein. Tau protein (eg, Genbank Accession Nos. NP_058519.3, NM_016835.5 and isoforms thereof) regulates microtubule dynamics and is associated with many diseases, including Alzheimer's disease.

[0042] As used herein, phosphorylated Tau 181 refers to tau protein phosphorylated on Threonine 181 of human Tau protein.

[0043] As used herein, phosphorylated Tau 217 refers to tau protein phosphorylated on Threonine 217 of human Tau protein.

[0044] As used herein, phosphorylated Tau 231 refers to tau protein phosphorylated on threonine 217 of human Tau protein.

[0045] As used herein, phosphorylated Tau 235 refers to tau protein phosphorylated on threonine 217 of human Tau protein.

[0046] As used herein, β-amyloid 42 refers to the cleavage product of 42 amino acids of amyloid β precursor protein. Amyloid β precursor protein (e.g., Genbank accession number P05067.3, NM_000484.4 and its subtypes) is a single transmembrane protein, expressed at high levels in the brain, and its function is unclear. It is proteolytically digested into many cleavage products, including β-amyloid 42 and β-amyloid 40, both of which are known to aggregate in plaques in patients with Alzheimer's disease.

[0047] As used herein, β-amyloid 40 refers to the cleavage product of 40 amino acids of the amyloid β precursor protein.

[0048] As used herein, the term "subject" includes people and animals that can suffer from or suffer from the dementia associated with CNS disorders, and the CNS disorders include neurodegenerative diseases, such as Alzheimer's disease, or any disorders directly or indirectly related to Alzheimer's disease. The example of the subject includes mammals, such as people, non-human primates, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats and transgenic non-human animals. In certain embodiments, the subject is a person, such as suffering from, having a risk of suffering from or potentially being able to suffer from Alzheimer's disease or Alzheimer's disease related dementia. In embodiments, the subject is aged about 45-50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old or greater than 90 years old.

[0049] As used herein, a "CDR" score refers to a score generated using the Clinical Dementia Rating Assessment Program developed by the Washington University Medical School (see Morris, CJ, Neurology, 1993; 43: 2412-2414). People who are determined to have no dementia (i.e., no detectable dementia) are designated as "0"; very mild dementia = 0.5; mild dementia = 1; moderate dementia = 2; severe dementia = 3. Individuals diagnosed with possible / probable Alzheimer's type dementia (DAT) are typically CDR 1 or higher. In the early stages (CDR 0.5, usually lasting 2-5 years or longer), most individuals meet the clinical criteria for mild cognitive impairment (MCI) (Peterson et al., Arch. Neurol, 1999; 56: 303).

[0050] As used herein, the Mini-Mental State Examination ("MMSE") score refers to a score generated using the MMSE protocol for evaluating cognitive therapy. As used herein, patients with an MMSE score of 27-30 are considered to have no cognitive impairment, patients with an MMSE score of 21-26 are considered to have mild cognitive impairment, patients with an MMSE score of 11-20 are considered to have moderate cognitive impairment, and patients with an MMSE score of 0-10 are considered to have severe cognitive impairment.

[0051] As used herein, a "level" of a biomarker in an individual without Alzheimer's disease includes, for example, a level of the biomarker in an individual that is representative of the average level of the biomarker in a population of individuals without Alzheimer's disease. Thus, the level in an individual without Alzheimer's disease can be determined by determining, for example, the average level of the biomarker in a population of individuals without Alzheimer's disease.

[0052] As used herein, a biomarker "level in an individual not at risk for developing Alzheimer's disease" includes, for example, a biomarker level in an individual that represents the average level of the biomarker in a population of individuals not at risk for developing Alzheimer's disease. Thus, the level in an individual not at risk for developing Alzheimer's disease can be determined by determining, for example, the average level of the biomarker in a population of individuals not at risk for developing Alzheimer's disease.

[0053] As used herein, a "sample" consists of biological material isolated from a subject, including but not limited to blood, serum, tissue, plasma, or cerebrospinal fluid. Certain embodiments provide methods for collecting samples (eg, blood and other materials) that can be used for diagnostic purposes.

[0054] In some embodiments, the sample or biological sample according to the present disclosure contains a cell population or cell fragments, including but not limited to cell membrane components, exosomes and subcellular components. These cells can be homogeneous cell populations, such as isolated cells of a specific type, or a mixture of different cell types, such as biological fluids or tissues from human or mammal or other species subjects. Other samples used for the method and for use with the composition include but are not limited to blood samples, including serum, plasma, whole blood and peripheral blood; saliva, urine, vaginal or cervical secretions, amniotic fluid, placental fluid, cerebrospinal fluid or serous fluid, mucosal secretions (e.g., oral, vaginal or rectal mucosal secretions). Other samples include blood-derived or biopsy-derived tissue biological samples or cell lysates (i.e., mixtures of tissue and / or cell sources). Other suitable tissues include hair, nails, etc. Other samples include antibody libraries, antibody fragments and antibody mimetics (such as affibodies). Such samples can be further diluted with saline, buffer or physiologically acceptable diluents. Alternatively, such samples are concentrated by conventional methods. Still other samples may be collections of synthetic or engineered chemical molecules, proteins, antibodies, or any other targets described herein.A sample is typically obtained or derived from a particular source, subject, or patient.

[0055] In a preferred embodiment, the biological sample used to determine the level of one or more biomarkers is a sample containing circulating biomarkers (e.g., extracellular biomarkers). Extracellular biomarkers circulate freely in various biological materials, including body fluids, such as liquids from the circulatory system, such as blood samples or lymph samples, or from other body fluids, such as CSF, urine, or saliva. Therefore, in some embodiments, the biological sample used to determine the level of one or more biomarkers is a body fluid, such as blood, its parts, serum, plasma, urine, saliva, tears, sweat, semen, vaginal secretions, lymphatic fluid, bronchial secretions, CSF, etc. In some embodiments, the sample is a sample obtained non-invasively. In some embodiments, the sample is obtained from a body fluid other than CSF.

[0056] Diagnosis of Alzheimer's disease

[0057] In some embodiments, the level of a biomarker (e.g., one or more biomarkers) is used to determine whether a subject (e.g., a patient) suffers from a neurodegenerative disease or disorder, such as Alzheimer's disease. In an embodiment, the level of a biomarker in a subject sample is compared to the normal level of the biomarker in one or more subjects without Alzheimer's disease, or compared to a reference standard. The normal level of a biomarker in a sample, or a reference standard, can be the average level of a biomarker in a sample of one or more healthy subjects (e.g., a subject with a CDR score of 0, such as a subject in the same age group, and optionally the same sex and / or race).

[0058] In an embodiment, the biomarkers are genes and / or phosphorylated proteins selected from those listed in Table 2.

[0059] In an embodiment, the biomarker is ACACA, AKT1, PRKAA1, ARAF, BAD, CASP9, CDC25A, CDK6, JUN, MYC, RAF-1, CCND1, CCNE1, EEF2, EEF2K, EIF2S1, EIF4B, EIF4E, MAPK3, FOXO3, FYN, GSK3A, GSK3B, ERBB2, NFKBIA, JAK1, JAK2, MAPK8, MTOR, RELA, MAPK14, MAPK11, MAPK13, MAPK12, TP53, RPS6KB1, PIK3R3, PPARA, PPARG, AKT1S1, PTEN, RB1, RPS6KA1, RPS6KA2, SGK3, SRC, STAT1, STAT3, MAPT, TBK1, TSC2, ZAP70, EGR1, IGF1R, IRS1, ESR1.

[0060] In embodiments, expression of a biomarker is increased compared to a control or reference standard, including, for example, the level of the biomarker in a subject without a neurodegenerative disease or disorder, such as Alzheimer's disease.

[0061] In embodiments, the phosphorylation level of a biomarker (e.g., a biomarker listed in Table 2) is increased or decreased compared to a control or reference standard (including, for example, the level of the biomarker in a subject without a neurodegenerative disease or disorder such as Alzheimer's disease). In some embodiments, the biomarker is selected from the group consisting of: acetyl-CoA carboxylase, Akt (pan)-phosphorylation, Akt (pan)-phosphorylation, Akt (pan)-phosphorylation, Akt1-phosphorylation, Akt1-phosphorylation, Akt2-phosphorylation, AMPKα-phosphorylation, A-Raf-phosphorylation, Bad-phosphorylation, caspase 9-phosphorylation, cdc25A (M phase inducer), CDK6-phosphorylation, c-Jun-phosphorylation, c-Jun-phosphorylation, c-Myc-phosphorylation, c-Raf-phosphorylation, cyclin D1-phosphorylation, cyclin E1-phosphorylation, eEF2-phosphorylation, eEF2k-phosphorylation, eIF2α-phosphorylation, eIF4B-phosphorylation, eIF4B-phosphorylation, eIF4E-phosphorylation, Erk1 / 2 (MAPK p44 / 42), Erk1 / 2 (MAPK p44 / 42), FoxO3a-phosphorylation, FoxO3a-phosphorylation, Fyn-phosphorylation, GSK3α / β-phosphorylation, GSK3α / β-phosphorylation, GSK3β-phosphorylation, Her2(ErbB2)-phosphorylation, IκBα-phosphorylation, Jak1-phosphorylation, Jak2-phosphorylation, JNKJSAPK-phosphorylation, mTOR phosphorylation, NF-κB p65-phosphorylation, p38 MAPK phosphorylation, p38 MAPKβ, p38 MAPKδ, p38 MAPKγ, p53-phosphorylation, p70 S6 kinase-phosphorylation, p70 S6 kinase-phosphorylation, P13 kinase p85 / p55-phosphorylation, PPARα-phosphorylation, PPARγ-phosphorylation, PRAS40-phosphorylation, PTEN-phosphorylation, Rb-phosphorylation, RSK 1-phosphorylation, RSK 3-phosphorylation, SGK3-phosphorylation, Src-phosphorylation, Src family-phosphorylation, STAT 1-phosphorylation, STAT 3-phosphorylation, Tau-phosphorylation, TBK1 (NAK)-phosphorylation, TSC2 (potato globulin)-phosphorylation, zap 70-phosphorylation, EGR1, IGF-1Rβ / IRβ-phosphorylation, IRS-1-phosphorylation, and ERα (estrogen receptor α)-phosphorylation.

[0062] In other embodiments, the normal level of a biomarker in a sample can be the average level of a biomarker in one or more healthy subject samples, and the Mini-Mental State Examination ("MMSE") scores of these subjects are between 27 and 30, such as subjects in the same age group, and optionally the same gender. During the MMSE, a doctor or other medical professional asks a series of questions to the patient, which are intended to test a series of daily mental skills. Frequently asked questions include, for example, remembering and repeating the names of three common objects, saying the year, date, season and day of the week, counting down from 100 in increments of 7, spelling the word "world" backwards, saying its name when the examiner points to a familiar object, identifying the location of the examiner's office, repeating the common phrase after the examiner states the common phrase, copying a picture of two interlocking shapes, and following a three-part series of instructions (e.g., picking up a piece of paper, folding it in half, and placing it on the floor). The maximum score for the MMSE examination is 30 points. In general, patients with an MMSE score of 27-30 are considered to have no cognitive impairment, patients with an MMSE score of 21-26 are considered to have mild cognitive impairment, patients with an MMSE score of 11-20 are considered to have moderate cognitive impairment, and patients with an MMSE score of 0-10 are considered to have severe cognitive impairment. In certain embodiments, patients with an MMSE score of 0-16 are considered to have late-stage (moderately severe to severe) Alzheimer's disease.

[0063] In one embodiment, the difference in biomarker levels is an increase relative to a normal control sample. Suitable controls can also be reference standards. Reference standards are used as reference levels for comparison so that samples from a subject can be compared with the reference standards to infer the Alzheimer's disease state of the subject. Reference standards can represent the level of one or more biomarkers in a known subject (e.g., a subject known to be a normal subject, or a subject known to suffer from Alzheimer's disease).

[0064] Likewise, a reference standard can represent the level of one or more biomarkers in a known subject population (e.g., a subject population known to be normal subjects, or a subject population known to have Alzheimer's disease). For example, a reference standard can be obtained by combining samples from multiple individuals and determining the level of one or more biomarkers in the combined samples, thereby generating a standard in an average population. Such a reference standard represents the average level of biomarkers in a population of individuals.

[0065] For example, a reference standard can also be obtained by averaging the levels of a biomarker determined to be present in individual samples obtained from multiple individuals. Such a standard also represents the average level of the biomarker in the individual population.

[0066] A reference standard can also be a set of values, each value representing a biomarker level for a known subject in a population of individuals. In certain embodiments, a test sample can be compared to such a set of values ​​to infer the Alzheimer's disease state of a subject.

[0067] In certain embodiments, the reference standard is an absolute value. In such embodiments, the test sample can be compared with the absolute value to infer the Alzheimer's disease state of the subject. In one embodiment, the level of one or more biomarkers in the sample is compared relative to a suitable control by executing a software classification algorithm. The technician can easily envision additional suitable controls, which may be suitable, depending on the determination in question. The above-mentioned suitable controls are exemplary and are not intended to be limiting.

[0068] Compared to normal subjects, subjects with elevated levels of one or more biomarkers may have Alzheimer's disease, including early Alzheimer's disease, moderate or intermediate Alzheimer's disease, or severe or late Alzheimer's disease. In one embodiment, the level of one or more biomarkers can be used to diagnose Alzheimer's disease in subjects with symptoms characteristic of early Alzheimer's disease (also known as prodromal Alzheimer's disease).

[0069] In another embodiment, the levels of one or more biomarkers can be used to diagnose Alzheimer's disease in a subject with symptoms characteristic of "moderate to severe cognitive decline" (also called "moderate" or "mid-stage" Alzheimer's disease). Moderate to severe cognitive decline is characterized by significant gaps in memory and the presence of deficits in cognitive function. At this stage, some assistance with daily activities is required.

[0070] In another embodiment, the levels of one or more biomarkers can be used to diagnose Alzheimer's disease in subjects with symptoms characteristic of "severe cognitive decline" (also known as "moderate" or "intermediate" Alzheimer's disease). In severe cognitive decline, memory difficulties continue to worsen, significant personality changes may occur, and affected individuals often require extensive assistance with routine daily activities.

[0071] Treatment of Alzheimer's disease

[0072] In some embodiments, an Alzheimer's disease treatment is administered following detection of an elevated level of a biomarker compared to a normal level of the biomarker.

[0073] As used herein, Alzheimer's disease treatment includes prescribing or administering one or more therapeutic interventions to slow, prevent, reverse, or modify the progression of the disease. Alzheimer's disease treatments can be drug or non-drug treatments. In some embodiments, Alzheimer's disease treatments or therapeutic agents can treat one or more symptoms of the disease. For example, a therapeutic intervention can include administering to a subject a therapeutically effective amount of at least one Alzheimer's disease therapeutic drug. In certain embodiments, an Alzheimer's disease therapeutic agent can be (Galantamine), (Rivastigmine), (donepezil), (memantine), or a pharmaceutically acceptable salt or ester thereof. In a specific embodiment, the treatment of Alzheimer's disease according to the present disclosure comprises administering therapeutic aducanumab (Aduhelm TM In some embodiments, Alzheimer's disease treatment comprises administration of Suvorexant ( ), Citalopram ), Fluoxetine ), Paroxetine ), Sertraline, ), Trazodone ), Lorazepam ), Oxazepam, ), Aripiprazole ), Clozapine ), Haloperidol ), Olanzapine ), Quetiapine ), Risperidone ), Ziprasidone ) or carbamazepine ( In other embodiments, Alzheimer's disease treatment may include non-drug treatment options, such as behavioral treatment options.

[0074] Pharmaceutical composition can be used to apply Alzheimer's disease therapeutic agent to subject.Suitable pharmaceutical composition includes Alzheimer's disease therapeutic agent (or its pharmaceutically acceptable salt or ester), and optionally includes a pharmaceutically acceptable carrier (carrier), suitable pharmaceutical composition such as including galanthamine, rivastigmine, donepezil or the pharmaceutical composition of the pharmaceutically acceptable salt or ester (such as galanthamine hydrobromide, rivastigmine tartrate, donepezil hydrochloride) of any of the foregoing.In certain embodiments, these compositions optionally further include one or more other therapeutic agents.

[0075] As used herein, the term "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio.

[0076] Pharmaceutically acceptable salts of amines, carboxylic acids and other types of compounds are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977) (incorporated herein by reference). Salts can be prepared in situ during the final separation and purification of the compounds of the present invention, or separately by reacting free base or free acid functional groups with suitable reagents. For example, free base functional groups can be reacted with suitable acids. In addition, when the compound carries an acidic portion, its suitable pharmaceutically acceptable salts may include metal salts, such as alkali metal salts, such as sodium or potassium salts; and alkaline earth metal salts, such as calcium or magnesium salts.

[0077] As used herein, the term "pharmaceutically acceptable ester" refers to an ester that hydrolyzes in vivo, and includes those esters that are easily decomposed in the human body to leave the parent compound or its salt. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic acids, alkenoic acids, cycloalkanoic acids and alkandioic acids, wherein each alkyl or alkenyl moiety advantageously has no more than 6 carbon atoms. As described above, the pharmaceutical composition may additionally include a pharmaceutically acceptable carrier. The term carrier includes any and all solvents, diluents or other liquid carriers (vehicles), dispersants or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., which are suitable for preparing the desired specific dosage form. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for preparing pharmaceutical compositions and known techniques for preparing them. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; the following may also be present in the composition, according to the judgment of the formulator: ethanol and phosphate buffer solutions, and other nontoxic and compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.

[0078] The terms "treat" or "treating" are used herein to refer to relieving, reducing or alleviating at least one symptom of a disease in a subject. For example, with respect to Alzheimer's disease, the term "treat" includes alleviating, reducing or alleviating cognitive impairment (such as memory and / or disorientation) or global dysfunction (overall function, including daily living activities) and / or slowing or reversing the gradual deterioration of global or cognitive impairment. Thus, the term "treat" also encompasses delaying or preventing the onset of disease and / or reducing the risk of developing or worsening disease symptoms before clinical manifestations of the disease or symptoms of the disease appear.

[0079] In some embodiments, after administering the Alzheimer's disease treatment, the level of the biomarker is monitored and, optionally, the level of the biomarker is compared to a normal level of the biomarker. In some embodiments, after administering a first dose of the Alzheimer's disease treatment, the level of the biomarker is determined, and if the level of the biomarker increases above the normal level, a second Alzheimer's disease treatment is administered at a dose higher than the first dose (e.g., 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times higher).

[0080] Detection Methods

[0081] In certain embodiments, mass spectrometry is used to detect the level of one or more biomarkers in a sample. In a specific embodiment, selected reaction monitoring mass spectrometry (SRM-MS) is used to detect the level of biomarkers. In other embodiments, other quantitative mass spectrometry techniques are used to detect the level of one or more biomarkers in a sample, including but not limited to spectral counting, isobaric mass labeling or ion mobility mass spectrometry.

[0082] In further embodiments, the absolute concentration of the one or more biomarkers is determined.In some embodiments, the absolute concentration of the one or more biomarkers is determined using a combination of SRM-MS and AQUA methods.

[0083] In other embodiments, the level of one or more biomarkers is determined by electrophoresis. For example, in some embodiments, the level of biomarkers is determined by, but not limited to, one-dimensional or two-dimensional electrophoresis or capillary electrophoresis. Those skilled in the art will recognize further quantitative electrophoresis methods suitable for practicing the present disclosure.

[0084] The level of a biomarker in a sample can be determined by an immunological method or an affinity-based method. For example, the level of a biomarker in a sample can be determined by evaluating (e.g., quantifying) the level of a biomarker in a sample, using, for example, immunohistochemical analysis, Western blot, ELISA, immunoprecipitation, flow cytometry analysis, or any other technique known in the art or described herein. In a specific embodiment, the level of a biomarker is determined by a method capable of quantifying the amount of a biomarker present in a patient sample and / or capable of detecting a correction of the level of a biomarker after treatment.

[0085] In some embodiments, the biomarkers of the present disclosure are contacted with a ligand having affinity for the biomarker. "Ligand" used in these compositions and methods refers to any naturally occurring or synthetic biological or chemical molecule that is used to specifically bind to the biomarker. Binding can be covalent or non-covalent, i.e., conjugated or combined by any known method, taking into account the properties of the ligand and its respective target. The ligand can be independently selected from a peptide, a protein, an antibody or an antibody fragment (e.g., an antigen-binding portion of an antibody), an antibody mimetic, an affibody, a ribonucleic acid sequence or a deoxyribonucleic acid sequence, an aptamer, a lipid, a polysaccharide, a lectin, or a chimeric molecule formed by a plurality of identical or different ligands. Additional non-limiting examples of ligands include Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv), bispecific antibodies (diabody, Dab), synbody, nanobody, BiTE, SMIP, DARPin, DNL, ​​Duocalin, adnectin, fynomer, Kunitz domain, Albu-dab, DART, DVD-IG, Covx-body, peptibody, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Hole, or combinations thereof. In some embodiments, the ligand is a recombinant protein or a naturally occurring protein. In certain embodiments, the ligand is a monoclonal antibody or a polyclonal antibody, or a fragment thereof. In some embodiments, the ligand of the construct can also be directly labeled with one or more detectable labels, such as fluorophores (see labels discussed below), which can be measured by methods independent of the measurement or detection of the polymer construct described elsewhere herein.

[0086] As used herein, the term "antibody" is intended to include fragments thereof, which also have specific reactivity with the subject polypeptide. Antibodies can be fragmented using conventional techniques, and the usefulness of the fragments can be screened in the same manner as described above for intact antibodies. For example, F(ab)2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab)2 fragments can be treated to reduce disulfide bonds to produce Fab fragments. Antibodies are further intended to include bispecific, single-chain, chimeric, humanized and fully human molecules, which have affinity for the biomarkers of the present disclosure, which is conferred by at least one CDR region of the antibody. The antibody may further include a label connected thereto and capable of being detected (e.g., the label may be a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor).

[0087] In certain embodiments, the antibody is a recombinant antibody, the term covering any antibody generated in part by molecular biology techniques, including CDR-grafted antibodies or chimeric antibodies, human or other antibodies assembled from antibody domains selected from a library, single-chain antibodies, and single-domain antibodies (e.g., human VH proteins or camelid VHH proteins). In certain embodiments, the antibody may be a monoclonal antibody, and in certain embodiments. For example, a method for producing a monoclonal antibody that specifically binds to a biomarker disclosed herein may include administering to a mouse an amount of an immunogenic composition comprising an antigen polypeptide that effectively stimulates a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse, and fusing the antibody-producing cells with myeloma cells to obtain an antibody-producing hybridoma, and testing the antibody-producing hybridoma to identify a hybridoma that produces a monoclonal antibody that specifically binds to an antigen. Once obtained, the hybridoma can be proliferated in a cell culture, optionally under culture conditions where the hybridoma-derived cell produces a monoclonal antibody that specifically binds to an antigen. The monoclonal antibody can be purified from the cell culture.

[0088] In some embodiments, the phosphorylation-specific antibodies provided by the present disclosure are specifically reactive with the phosphorylated form of the biomarker. As used herein, "specifically reactive with..." with respect to an antibody is intended to indicate that, as generally understood in the art, the antibody is selective between an antigen of interest (e.g., a phosphorylated form of a biomarker) and other antigens of no interest (e.g., an unphosphorylated biomarker, or the total amount of a biomarker present in a sample). Monoclonal antibodies are generally more inclined (compared to polyclonal antibodies) to effectively distinguish between desired antigens and cross-reactive polypeptides. A feature that affects the specificity of antibody: antigen interactions is the affinity of the antibody for the antigen. Although the desired specificity can be achieved through a range of different affinities, in general, the affinity (dissociation constant) of the preferred antibody is about 1x10 -8 M or smaller.

[0089] In addition, the technique used to screen antibodies to identify the desired antibody may affect the properties of the antibody obtained. For example, if the antibody will be used to bind an antigen in solution, a test solution may be required for binding. There are a variety of different techniques that can be used to test the interaction between the antibody and the antigen to identify a particularly desired antibody. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore.TM. binding assays, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., IGEN International, Inc., Gaithersburg, Md.'s paramagnetic bead system), Western blotting, immunoprecipitation assays, and immunohistochemistry.

[0090] In one embodiment, the level of a biomarker in a tissue sample is determined by evaluating (e.g., quantifying) the protein expression of the biomarker in the sample using ELISA. For example, a sandwich ELISA method can be used to identify biomarkers in human serum and quantify them. A sandwich ELISA method for determining the level of a biomarker in a tissue sample can include coating an ELISA plate with one or more anti-biomarker antibodies, contacting the plate with a sample (e.g., human serum), and detecting the biomarker ligands in the sample.

[0091] Phosphorylation-specific antibodies for use in assays to measure biomarker levels in a sample are known in the art or can be readily developed using methods known to those skilled in the art. Table 1 provides examples of monoclonal antibodies that can be used in assays to measure biomarker levels according to the present disclosure.

[0092] Table 1 : Representative antibodies for biomarker determination

[0093]

[0094] In some embodiments, the ligand or antibody of the present disclosure further comprises a detectable label, which refers to a reagent, part or compound that can provide a detectable signal, depending on the assay format adopted. Labeling can be associated only with the ligand or antibody, or associated with a complex formed with the ligand or antibody and one or more features of the present disclosure. Such labels can provide detectable signals alone, or provide detectable signals in combination with other compositions or compounds. In one embodiment, the label has the required interactivity to produce detectable signals. In certain embodiments, the label is visually detectable, such as detectable by colorimetry. Various enzyme systems play a role in determining to reveal colorimetric signals, for example, glucose oxidase (which uses glucose as a substrate) releases peroxide as a product, which produces oxidized TMB in the presence of peroxidase and a hydrogen donor (such as tetramethylbenzidine (TMB)), which can be seen as blue. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in combination with glucose-6-phosphate dehydrogenase, which reacts with ATP, glucose and NAD+, producing NADH in particular, among other products, which is detected as an increase in absorbance at a wavelength of 340 nm. Other labeling systems that can be used in the described methods and constructs can be detected by other means, for example, colored latex particles (Bangs Laboratories, Indiana) embedded with dyes can be used instead of enzymes to provide visual signals indicating the presence of labeled ligands or antibodies in applicable assays. Other labels include fluorescent compounds, fluorophores, radioactive compounds or elements. In one embodiment, fluorescence detectable fluorescent dyes such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 or -7 (PC5 or PC7), PE-Texas Red (PE-Texas Red, ECD), PE-cyanin-5.5, rhodamine, PerCP and Alexa dyes. Combinations of such labels may be used, particularly such as Texas Red and rhodamine, FITC+PE, FITC+PECy5 and PE+PECy7, depending on the assay method. Given the present specification, it is within the skill of the art to select and / or generate suitable labels for labeling any component of the ligand and / or ligand or antibody of the present disclosure.

[0095] 1. Quantitative immuno-PCR

[0096] In embodiments, the biomarkers of the present disclosure are detected by quantitative immuno-PCR (qIPCR) assays.Thus, in certain embodiments, the biomarkers of the present disclosure are detected by ligands, such as antibodies linked to oligonucleotides with primer sites and a barcode unique to the ligand.

[0097] As used herein, the term "attachment" or "attachment" describes the interaction between the components of a construct and refers to covalent attachment or various non-covalent types of attachment. Other attachment chemistries that can be used to assemble the constructs described herein include, but are not limited to, sulfhydryl-maleimide, sulfhydryl-haloacetate, amine-NHS, amine-isothiocyanate, azide-alkyne (CuAAC), tetrazole-cyclooctene (iEDDA) (see, e.g., reference 24 and other references therein). In one embodiment, each polymer construct is connected to a ligand by an irreversible covalent bond. In another embodiment, each polymer construct is connected to a ligand by a cleavable covalent bond (e.g., a disulfide bond or a photocleavable linker).

[0098] Therefore, in some embodiments, a DNA, RNA, DNA / RNA hybrid, or a fragment, analog or derivative thereof used as a marker is specifically linked to an antigen-antibody complex using a linker molecule having a bispecific binding affinity for nucleic acids and antibodies, resulting in the formation of an antigen-antibody-linker-DNA conjugate. A segment of the linked marker is enzymatically amplified (e.g., by polymerase chain reaction with suitable primers). The presence of a specific product of a polymerase chain reaction or other amplification method indicates that the marker molecule is specifically linked to the antigen-antibody complex, and accordingly, this indicates the presence of an antigen.

[0099] The ligand may be linked to the 5' end or any other part of the construct oligonucleotide sequence, provided that the linking or conjugation does not prevent the function of the components of the construct oligonucleotide sequence.

[0100] Generally, oligonucleotide can be any length, and this length adapts to the length of its functional component.In one embodiment, the length of oligonucleotide is between 20 and 100 monomer components (such as nucleic acid base).In some embodiments, the length of oligonucleotide is at least 20,30,40,50,60,70,80,90 or more than 100 monomer components (such as nucleic acid base).In other embodiments, the length of oligonucleotide is 200 to about 400 monomer components (such as nucleotide).

[0101] In certain embodiments, the polymer construct is generally composed of deoxyribonucleic acid (DNA). In some embodiments, the oligonucleotide is a DNA sequence. In other embodiments, the oligonucleotide or part thereof comprises a modified DNA base. The modification of the DNA base is known in the art and may include chemically modified bases, including labels.

[0102] In other embodiments, the oligonucleotide or a portion thereof comprises a ribonucleic acid (RNA) sequence or a modified ribonucleotide base. Modification of RNA bases is known in the art and may include chemically modified bases, including labels. In other embodiments, different portions of the oligonucleotide sequence may include DNA and RNA, modified bases, or modified polymer connections (including but not limited to PNA and LNA).

[0103] As used herein, the term "primer sequence" refers to the functional components of a construct oligonucleotide sequence, which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for the amplification of an oligonucleotide sequence. The primer sequence can be formed by a polymer of a combination of DNA, RNA, PNA, modified bases or these bases or polyamides, etc. In one embodiment, the length of the primer sequence is about 10 such monomer components (e.g., nucleotide bases). In other embodiments, the length of the primer sequence is at least about 5 to 100 monomer components (e.g., nucleotides). Thus, in various embodiments, the primer sequence consists of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 sequences of monomer components (e.g., nucleic acids). In certain embodiments, for example, in a multiplexed biomarker assay according to the present disclosure, multiple oligonucleotide sequences are linked to multiple ligands (e.g., antibodies), and the primer sequences may be the same or different, depending on the technique intended for amplification. In certain embodiments, the primer sequence may be a universal sequence suitable as an annealing site for various amplification techniques.

[0104] Amplification techniques include, but are not limited to, DNA polymerase-based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and amplification by any number of DNA polymerases (e.g., T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase); and RNA polymerase-based amplification systems (e.g., T7-, T3-, and SP6-RNA polymerase amplification), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), branched amplification methods, and RNA-seq23.

[0105] As used herein, the term "barcode" describes a defined polymer, such as a polynucleotide, that is specific for a single ligand when it is a functional element of an oligonucleotide. As used in the various methods described herein, the term barcode can be a "cell barcode" or a "substrate barcode," which describes a defined polynucleotide that is used to specifically identify a specific cell or substrate (e.g., drop-seq beads). In either embodiment, the barcode can be formed by a defined sequence of DNA, RNA, modified bases, or a combination of these bases, and any other polymer defined above. In one embodiment, the length of the barcode is about 2 to 4 monomer components (e.g., nucleotide bases). In other embodiments, the length of the barcode is at least about 1 to 100 monomer components (e.g., nucleotides). Thus, in various embodiments, the barcode sequence consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 99, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomer components (e.g., nucleic acids) are formed.

[0106] In some embodiments, the oligonucleotides of the present disclosure further include an anchor sequence intended to hybridize with another oligonucleotide sequence (e.g., capture polymer, capture oligonucleotide, primer, etc.). In certain embodiments, an anchor is designed for the purpose of generating a double-stranded construct oligonucleotide sequence. In some embodiments, the anchor is located at the 3' end of an oligonucleotide sequence (e.g., a construct oligonucleotide sequence). In other embodiments, the anchor is located at the 5' end of a construct oligonucleotide sequence. In some embodiments, each anchor has specificity for its predetermined complementary sequence. For example, in certain embodiments, the anchor is configured to hybridize with the 3' end of the capture oligonucleotide so that the 3' end of the capture oligonucleotide serves as a primer that can generate a second complementary chain of an oligonucleotide in the presence of a polymerase. In certain embodiments, when a composition or method comprises a plurality of "first constructs", each first construct has the same anchor sequence. In some embodiments, each additional anchor has a different additional sequence that hybridizes with different complementary sequences. In other embodiments, each additional anchor may have the same anchor sequence as the first construct or other constructs, depending on the assay method step. When used in the various methods described herein, the anchor can hybridize with a free complementary sequence or with a complementary sequence fixed on a substrate. In certain embodiments, the anchor can be formed by a monomer sequence of a selected polymer, such as DNA, RNA, a modified base or a combination of these bases, PNA, polyamide, etc. In one embodiment, the length of the anchor is about 3 to 15 monomer components (e.g., nucleotides). In other embodiments, the length of each anchor can be at least about 3 to 100 monomer components (e.g., nucleotides). In some embodiments, the anchor comprises a monomer component (e.g., nucleotide) of 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15. In various embodiments, the anchor is composed of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 94, 95, 96, 97, 98, 99 or up to 100 monomer components (e.g., nucleic acids) are formed.

[0107] In some embodiments, the anchor sequence comprises or consists of a polyA sequence. In certain embodiments, the polyA sequence comprises a nucleic acid sequence comprising ten or more (e.g., 10-40, 10-30, or 10-20) consecutive adenosine nucleotides, derivatives or variants of adenosine nucleotides, etc., or a combination thereof. In other embodiments, the anchor sequence comprises or consists of a polyT sequence. In another embodiment, the anchor sequence is a polyG sequence. In other embodiments, the anchor sequence can be a random sequence, provided that it can hybridize with its predetermined complementary sequence (e.g., capture oligonucleotide, amplification primer, etc.). For example, in some embodiments, the methods described herein can utilize a variety of oligonucleotides (e.g., a variety of constructs, which contain ligands connected to oligonucleotides), some or all of which contain different anchors (i.e., anchors with different nucleic acid sequences, or anchors with substantially different nucleic acid sequences). In some embodiments, the methods described herein can utilize a variety of oligonucleotides (e.g., a variety of constructs, which contain ligands connected to oligonucleotides), some or all of which contain the same anchor. In some embodiments, the methods described herein can utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising ligands attached to the oligonucleotides), some or all of which comprise substantially identical anchors (e.g., comprising substantially identical nucleic acid sequences). In some embodiments, the methods described herein can utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising ligands attached to the oligonucleotides), some or all of which comprise anchors comprising polyA sequences. In some embodiments, the polyA sequences of the plurality of anchors are substantially identical. As will be appreciated by those skilled in the art, substantially identical polyA sequences may differ substantially in length. In some embodiments, the polyA sequence (e.g., the polyA sequence of the anchor) is a nucleic acid configured to hybridize with a polyT sequence (e.g., an oligonucleotide or capture oligonucleotide comprising a polyT sequence). As will be appreciated by those skilled in the art, depending on the hybridization conditions, the polyA sequence may comprise one, two, three, or four non-polyA nucleotides and still effectively hybridize with the polyT sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three, or more mismatches. Thus, in some embodiments, the polyA sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% adenosine nucleotides, adenosine analogs, adenosine variants, or combinations thereof.

[0108] In some embodiments, the oligonucleotide comprises a polyT sequence. In some embodiments, the capture oligonucleotide comprises a polyT sequence (e.g., a 3′ polyT sequence). In some embodiments, the methods described herein can utilize a variety of oligonucleotides (e.g., a variety of capture oligonucleotides), some or all of which comprise a polyT sequence. In some embodiments, the polyT sequences of a variety of oligonucleotides are substantially the same. In some embodiments, a variety of capture oligonucleotides (e.g., a variety of different capture oligonucleotides, e.g., different bead-specific capture oligonucleotides) comprise substantially the same polyT sequence. As will be appreciated by those skilled in the art, substantially the same polyT sequence can be substantially different in length. In some embodiments, the polyT sequence comprises a length of 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive nucleotides (e.g., nucleotide length). In certain embodiments, the polyT sequence comprises a nucleic acid sequence comprising three or more, ten or more, 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive thymidine nucleotides, derivatives or variants of thymidine nucleotides, etc., or combinations thereof. In some embodiments, the polyT sequence (e.g., the polyT sequence of the capture oligonucleotide) is a nucleic acid configured to hybridize with a polyA sequence. As will be appreciated by those skilled in the art, depending on the hybridization conditions, the polyT sequence may comprise one, two, three, or four non-thymidine nucleotides and still effectively hybridize with the polyA sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three, or more mismatches. Thus, in some embodiments, the polyT sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% thymidine nucleotides, thymidine analogs, thymidine variants, or combinations thereof. In some embodiments, the polyT sequence comprises one or more uracil nucleotides, or derivatives thereof.

[0109] In an embodiment, a "joint" includes any portion for connecting or associating a ligand with an oligonucleotide sequence. Thus in one embodiment, the joint is a covalent bond. In another embodiment, the joint is a non-covalent bond. In another embodiment, the joint is composed of at least one to about 25 atoms. Thus in various embodiments, the joint is formed by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 atoms. In yet another embodiment, the joint is at least one to about 60 nucleic acids. Thus, in various embodiments, the linker is formed by a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, up to 60 nucleic acids. In yet another embodiment, the linker refers to at least one to about 30 amino acids. Thus, in various embodiments, the linker is formed by a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, up to about 30 amino acids.

[0110] In other embodiments, the linker may be a larger compound or two or more compounds that are covalently or non-covalently associated. In other embodiments, the linker may be a combination of linkers as defined herein.

[0111] In some embodiments, the linker used in the present disclosure is cleavable. In other embodiments, the linker used in the present disclosure is non-cleavable. In certain embodiments, the linker is a cleavable linker, such as a disulfide bond or a photocleavable bond.

[0112] In certain exemplary embodiments, the linker comprises a complex in which biotin is bound to the construct oligonucleotide sequence via a disulfide bond, with streptavidin bound to a ligand (e.g., an antibody). In another embodiment, biotin is bound to the ligand, and streptavidin is bound to the construct oligonucleotide sequence. The linker can be covalently linked to any oligonucleotide sequence portion of the construct, or conjugated in a manner other than covalent. In yet another embodiment, when the ligand is a recombinant antibody or a synthetic antibody, the linker can be engineered into the antibody sequence to facilitate 1: 1 coupling with the polymer construct, thereby simplifying the manufacture of the ligand, construct, and / or polymer construct. For example, for such purposes, The linker is engineered into the selected ligand (e.g., antibody), or into the polymer construct or component. Additionally or alternatively, the ligand is linked to the polymer construct when produced in the same cell. See, e.g., Vector Systems Technical Manual (TM254-5 / 17 Revised, Copyright 2017 by Promega Corporation) Scheme; and Janssen D.B., "Evolving haloalkaline dehalogenase", Curr. Opin. Chem. Biol., 2004, 8: 150-159.

[0113] In some embodiments, streptavidin-protein A chimera is used as a linker molecule. The chimera has two independent specific binding abilities. One is its combination with biotin, which is derived from the streptavidin part, and the other is its combination with the Fc part of immunoglobulin G (IgG) molecule, which is derived from the protein A part. This bifunctional specificity to both biotin and antibody allows any biotinylated nucleic acid molecule to be specifically conjugated to the antigen-antibody complex. Other linker molecules can also be advantageously utilized, such as any protein, peptide, nucleic acid marker cross-linked with antibody chemistry, or biotinylated marker nucleic acid cross-linked with biotinylated antibody by streptavidin or avidin.

[0114] 2. Digital Droplet PCR

[0115] In some embodiments, the ligand-oligonucleotide-biomarker complex separated according to the methods disclosed herein is amplified by digital droplet PCR. Therefore, in embodiments, the oligonucleotide population according to the present disclosure can be encapsulated into a droplet. In some embodiments, the oligonucleotides are encapsulated at a relatively low concentration, such as so that the droplets contain less than one oligonucleotide per droplet on average. Therefore, most or all of the oligonucleotides are amplified, such as substantially uniformly amplified.

[0116] In some embodiments, multiple primers can be added to the droplets to cause amplification, for example, using droplet-based PCR or other techniques known to those of ordinary skill in the art. In some cases, there can be at least 3, at least 5, at least 10, at least 30, at least 50, at least 100, at least 300, at least 500, at least 1000, at least 2000, at least 3000, at least 5000, or at least 10000, or more distinguishable primers. This can help, for example, to ensure that a large number of potential target oligonucleotides are amplified.

[0117] In some embodiments, the oligonucleotides in the amplified droplets can be determined or sequenced, for example, using any of a variety of techniques. For example, in one set of embodiments, the droplets can be broken and their contents combined together, for example, to produce a pool of amplified oligonucleotides. The pool of amplified oligonucleotides can then be sequenced for quantitative determination, for example, using techniques such as Illumina sequencing, single molecule real-time sequencing (e.g., Pacbio sequencing), nanopore sequencing, etc. to sequence and determine their levels.

[0118] Reagent test kit

[0119] In another aspect, the invention provides kits for diagnosing Alzheimer's disease status in a subject, which can be used to determine the level of one or more of: phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid, and combinations thereof.

[0120] The kit may include materials and reagents suitable for selectively detecting the presence of a biomarker that is diagnostic of Alzheimer's disease in a sample derived from a subject. For example, in one embodiment, the kit may include one or more phosphorylation-specific ligands, such as one or more phosphorylation-specific antibodies, that are capable of diagnosing Alzheimer's disease in a sample derived from a subject. The kit may include reagents that can be used to perform an assay to detect one or more biomarkers, for example, reagents that can be used to detect phosphorylation-specific ligands that bind to a biomarker of the present disclosure.

[0121] In further embodiments, the kit may contain instructions for appropriate operating parameters in the form of a label or product insert. For example, the instructions may include information or instructions on how to collect a sample, how to determine the level of one or more biomarkers in a sample, or how to correlate the level of one or more biomarkers in a sample with the Alzheimer's disease status of a subject.

[0122] In another embodiment, the kit may contain one or more containers containing miRNA biomarker samples to be used as reference standards, appropriate controls, or for calibration of assays to detect miRNA biomarkers in test samples.

[0123] A computer system having one or more processors and a memory is also provided, the memory storing one or more programs, which are executed by the one or more processors. Such a system includes a memory storing instructions for causing the computer system to perform any method described herein, including a portion of any method disclosed herein.

[0124] The features of the embodiments described herein can be implemented, used or implemented, used by means of a computer program product, such as a storage medium or a computer-readable storage medium, on which / the instructions stored therein can be used for programming a processing system to perform any feature displayed herein. Storage media (e.g., memory) can include, but are not limited to, high-speed random access memory, such as DRAM, SRAM, DDR RAM or other random access solid-state memory devices, and can include non-volatile memory, such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices or other non-volatile solid-state storage devices. In some embodiments, memory can include one or more storage devices away from the CPU. Memory (or the non-volatile memory device in these memories) includes a non-temporary computer-readable storage medium.

[0125] Describe the subject technology in terms of terms

[0126] For convenience, various examples of aspects of the present disclosure are described as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the subject technology. The figure identifiers and reference numbers provided below are only for example and for illustrative purposes, and these clauses are not limited by these identifiers.

[0127] Clause 1. A method of diagnosing and treating Alzheimer's disease in a subject in need thereof, the method comprising:

[0128] determining the presence of one or more biomarkers in a sample collected from a subject,

[0129] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and

[0130] If the one or more biomarkers are detected in the sample, an Alzheimer's disease treatment is administered.

[0131] Clause 2. A method of selecting and treating Alzheimer's disease in a subject in need thereof, the method comprising:

[0132] selecting a subject with Alzheimer's disease in need of such treatment by determining the presence of one or more biomarkers in a sample collected from the subject,

[0133] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0134] wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and

[0135] If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

[0136] Clause 3. A method of identifying and treating Alzheimer's disease in a subject in need thereof, the method comprising:

[0137] identifying a human subject with Alzheimer's disease in need of such treatment by determining the level of one or more biomarkers in a sample collected from the subject,

[0138] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0139] wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and

[0140] If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

[0141] Clause 4. A method of selecting one or more human subjects for treating Alzheimer's disease or alleviating a symptom thereof, the method comprising:

[0142] determining the level of one or more biomarkers in samples collected from one or more human subjects;

[0143] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0144] If the one or more biomarkers are detected in the sample, selecting the subject for treatment, and

[0145] Optionally, an Alzheimer's disease treatment is administered.

[0146] Clause 5. A method of diagnosing and treating a subject at risk of developing Alzheimer's disease, the method comprising,

[0147] determining the level of one or more biomarkers in a sample collected from the subject,

[0148] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0149] wherein detection of the one or more biomarkers in the sample indicates that the subject is at risk for developing Alzheimer's disease, and

[0150] If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

[0151] Clause 6. A method of diagnosing Alzheimer's disease in a subject, the method comprising:

[0152] determining the level of one or more biomarkers in a sample collected from the subject,

[0153] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0154] If the one or more biomarkers are detected, Alzheimer's disease is diagnosed in the subject.

[0155] Clause 7. A method for determining whether a subject is at risk for developing Alzheimer's disease, the method comprising,

[0156] determining the level of one or more biomarkers in a sample collected from the subject,

[0157] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0158] wherein detection of the one or more biomarkers indicates that the subject is at risk for developing Alzheimer's disease.

[0159] Clause 8. A method of diagnosing and treating Alzheimer's disease or alleviating a symptom thereof in a human subject, the method comprising:

[0160] determining an increased level of one or more biomarkers in a sample collected from the subject,

[0161] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0162] wherein the increased levels are measured compared to the levels of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease, and

[0163] If an increased level of the one or more biomarkers is detected in the sample, an Alzheimer's disease treatment is administered.

[0164] Clause 9. A method of selecting and treating a subject suffering from Alzheimer's disease, the method comprising:

[0165] selecting a human subject having Alzheimer's disease by determining an increased level of one or more biomarkers in a sample collected from the subject,

[0166] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, and

[0167] If an increased level of the one or more biomarkers is detected in the sample, administering an Alzheimer's disease treatment,

[0168] wherein the increased levels are measured as compared to the levels of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease.

[0169] Clause 10. A method of identifying and treating a subject suffering from Alzheimer's disease, the method comprising:

[0170] identifying a human subject having Alzheimer's disease by determining an increased level of one or more biomarkers in a sample collected from the subject,

[0171] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0172] If an increased level of the one or more biomarkers is detected in the sample, administering an Alzheimer's disease treatment,

[0173] wherein the increase is measured as compared to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease.

[0174] Clause 11. A method of selecting a subject in need of treatment for Alzheimer's disease, the method comprising:

[0175] determining the level of one or more biomarkers in a sample collected from the subject,

[0176] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, and

[0177] comparing the level of the one or more biomarkers in the sample to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease,

[0178] selecting the subject if the level of the one or more biomarkers in the sample is increased compared to the level in an individual without Alzheimer's disease, and

[0179] Optionally, an Alzheimer's disease treatment is administered to the subject.

[0180] Clause 12. A method of treating a subject at risk for developing Alzheimer's disease, the method comprising,

[0181] determining the level of one or more biomarkers in a sample collected from the subject,

[0182] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0183] comparing the level of the one or more biomarkers to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual not at risk for developing Alzheimer's disease, and

[0184] If the one or more biomarkers are increased in the subject relative to individuals not at risk for developing Alzheimer's disease, an Alzheimer's disease treatment is administered.

[0185] Clause 13. A method of measuring the effectiveness of a treatment for Alzheimer's disease, the method comprising:

[0186] To identify subjects with a CDR score greater than 0 or an MMSE score less than 27,

[0187] determining pre-treatment levels of one or more biomarkers in a sample collected from the subject,

[0188] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0189] administering Alzheimer's disease treatment, and

[0190] determining the post-treatment level of one or more biomarkers selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase in a second sample collected from the subject,

[0191] Wherein a decrease in the post-treatment level of the biomarker in the second sample relative to the first sample indicates effectiveness of the treatment.

[0192] Clause 14. A method for diagnosing Alzheimer's disease, the method comprising:

[0193] determining the level of one or more biomarkers in a sample collected from the subject,

[0194] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0195] comparing the level of the one or more biomarkers in the sample to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual without Alzheimer's disease, and

[0196] If the one or more biomarkers are increased in the subject relative to individuals without Alzheimer's disease, Alzheimer's disease is diagnosed in the subject.

[0197] Clause 16. A method for determining whether a subject is at risk for developing Alzheimer's disease, the method comprising,

[0198] determining the level of one or more biomarkers in a sample collected from the subject,

[0199] wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase,

[0200] comparing the level of the one or more biomarkers in the sample to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual not at risk for developing Alzheimer's disease, and

[0201] If the one or more biomarkers are increased in the sample relative to individuals not at risk for developing Alzheimer's disease, the subject is determined to be at risk for developing Alzheimer's disease.

[0202] Clause 17. The method according to any one of clauses 1-15, wherein the one or more biomarkers further comprise phosphorylated Tau protein and / or β-amyloid protein.

[0203] Item 18. The method according to Item 16, wherein the phosphorylated Tau protein is phosphorylated Tau181, phosphorylated Tau 217, phosphorylated Tau 231 and / or phosphorylated Tau 235.

[0204] Clause 19. The method of clause 17, wherein the β-amyloid proteins are β-amyloid 40 and β-amyloid 42, and wherein the ratio of β-amyloid 42 to β-amyloid 40 is increased relative to a subject having a CDR of 0 or an MMSE score of about 27-30.

[0205] Clause 20. The method of any one of Clauses 8-11 or Clause 14, wherein the individual without Alzheimer's disease has a CDR of 0 or an MMSE score of about 27-30.

[0206] Clause 21. The method according to any one of clauses 1-19, wherein the level of the one or more biomarkers is determined using antibodies specific for the one or more biomarkers.

[0207] Clause 22. The method according to Clause 20, wherein the level of the one or more biomarkers is determined by immunohistochemical analysis, Western blot, ELISA, immunoprecipitation and / or flow cytometric analysis.

[0208] Clause 23. The method according to clause 20, further comprising using quantitative immuno-PCR to detect the level of one or more biomarkers, comprising:

[0209] contacting the sample with a ligand linked to an oligonucleotide specific for the one or more biomarkers,

[0210] wherein the oligonucleotide comprises a primer sequence unique to the biomarker,

[0211] allowing the ligand to bind to the biomarker in the sample to form a complex comprising the ligand, the biomarker, and the oligonucleotide,

[0212] Isolation of the complex, and

[0213] Amplification oligonucleotides.

[0214] Item 24. The method according to Item 22, wherein the ligand and oligonucleotide are non-covalently linked via a linker.

[0215] Clause 25. The method according to Clause 23, wherein the ligand is an antibody, the oligonucleotide is biotinylated, and wherein the linker comprises a streptavidin-protein A chimera.

[0216] Item 26. The method according to Item 22, wherein the oligonucleotide is amplified by RT-PCR.

[0217] Clause 27. The method according to clause 22, wherein the oligonucleotides are amplified by digital droplet PCR.

[0218] Clause 28. The method of clause 22, wherein the oligonucleotide further comprises a unique barcode identifying the oligonucleotide, and wherein the oligonucleotide is sequenced to identify the attached ligand.

[0219] Clause 29. The method according to any one of clauses 1-16, wherein the levels of at least two of the following are detected: phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235 and B-amyloid.

[0220] Clause 30. The method according to any one of clauses 1-5 and 8-13, wherein the treatment comprises administering to the subject one or more of: (Galantamine), (Rivastigmine), (donepezil), (memantine), or a pharmaceutically acceptable salt or ester thereof.

[0221] Clause 31. The method according to any one of clauses 1-5 and 8-13, wherein the treatment comprises administering to the subject Aducanumab TM ).

[0222] Clause 32. The method according to any one of clauses 1 to 30, wherein the sample is blood, serum, tissue, plasma or cerebrospinal fluid.

[0223] Clause 33. A method of diagnosing and treating Alzheimer's disease or alleviating a symptom thereof in a human subject, the method comprising:

[0224] determining an increased level of one or more biomarkers in a sample collected from the subject,

[0225] wherein the one or more biomarkers are phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235 and B-amyloid protein,

[0226] wherein the increased levels are measured as compared to the levels of phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid in individuals without Alzheimer's disease, and

[0227] If the level of the one or more biomarkers in the sample is increased, an Alzheimer's disease treatment is optionally administered.

[0228] The present disclosure is further illustrated by the following examples, which are provided to aid in practicing the disclosed methods. These examples are not intended to limit the scope of the present disclosure in any way.

[0229] Example

[0230] Example 1 : Identification of expression and / or phosphorylation changes of certain genes and / or proteins in Alzheimer's disease models.

[0231] Neuronal cell line SH-SY5Y is obtained from European Collection of Authenticated Cell Culture (ECACC cat#EC94030304). SH-SY5Y cells are established in culture, amplified, and frozen in liquid nitrogen in freezing buffer, which consists of a culture medium containing 10% (v / v) dimethyl sulfoxide (DMSO). For example, SH-SY5Y cells are cultured in F12 / MEM culture medium supplemented with 15% heat-inactivated FBS, 1% non-essential amino acids, 50U / ml penicillin and 50μg / ml streptomycin and L-glutamine at 37°C, 5% CO2 atmosphere.

[0232] Amyloid β1-42 (Aβ1-42) was oligomerized by first dissolving Aβ1-42 (5 mM) in anhydrous DMSO and then further diluting it in ice-cold cell culture medium (Ham's F-12 without phenol red) to a final concentration of 100 μM. After vortexing for 30 seconds, the Aβ solution was incubated at 4°C for 24 hours. Aβ1-42 monomers were incubated at 4°C, physiological ionic strength and neutral pH to produce small-sized Aβ1-42 oligomeric assemblies.

[0233] SH-SY5Y cells were then removed from liquid nitrogen storage and seeded into collagen-coated 6-well cell culture plates. In order to avoid serum albumin binding to Aβ1-42, all experiments were performed in serum-free Neurobasal-A medium supplemented with B27. After 24 hours of culture, SH-SY5Y cells were first treated with 1 μM freshly prepared oligomeric Aβ1-42 for 24 hours (or 48 hours) and then treated with 100 μM hydrogen peroxide for 45 minutes. Untreated SH-SY5Y cells incubated for 45 minutes, 24 hours or 48 hours were used as controls. Bright field microscopy was used to record the viability of treated cells. At 45 minutes, 24 hours and 48 hours, the culture medium was removed and stored in 15 mL conical tubes at -80 ° C for future studies.

[0234] Subsequently, the treated SH-SY5Y cells were removed from the plate using a scraper. Next, the cells were pelleted by centrifugation in a 1.5 mL Eppendorf tube, the supernatant was removed and the pellet was frozen at -80 °C. The concentration and phosphorylation status of the 80 proteins listed in Table 2 were then analyzed by the AssayEngineers Hyperplex platform to identify proteins whose expression levels and / or phosphorylation status changed compared to the untreated control. Notably, the levels of phosphorylated FYN 416 and phosphorylated p38 MAP kinase were increased in the treated cells compared to the control cells.

[0235] Table 2 :Examining gene expression and phosphorylation changes through AssayEngineers' HyperPlex platform

[0236]

[0237]

[0238]

[0239] Example 2 : Case study.

[0240] An 85-year-old male presents to a geriatrician with memory loss. After initial evaluation, a preliminary diagnosis of dementia is made. The physician orders a blood test. Blood is drawn and serum fractions are separated. The purified serum lysate is incubated with phosphorylation-specific antibodies from a biomarker panel containing each of the following: phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and immuno-PCR is performed using digital droplet PCR.

[0241] An increased level of each phosphorylated biomarker was detected in the sample compared to the reference standard. Therefore, the subject was diagnosed with Alzheimer's disease. Therefore, the patient received a course of aducanumab and had fresh blood tested every 30 days for the biomarker panel.

[0242] Although the present disclosure is described and illustrated herein by reference to various specific materials, procedures and embodiments, it should be understood that the present disclosure is not limited to the specific combination of materials and procedures selected for this purpose. As will be appreciated by those skilled in the art, many variations of such details may be implied. This is intended to treat the specification and embodiments as merely exemplary, and the true scope and spirit of the present disclosure are indicated by the appended claims. All references, patents and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

1. A method for diagnosing and treating Alzheimer's disease in a subject in need thereof, the method comprising: determining the presence of one or more biomarkers in a sample collected from a subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and If the one or more biomarkers are detected in the sample, an Alzheimer's disease treatment is administered.

2. A method for selecting and treating Alzheimer's disease in a subject in need thereof, the method comprising: selecting a subject with Alzheimer's disease in need of such treatment by determining the presence of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

3. A method of identifying and treating Alzheimer's disease in a human subject in need thereof, the method comprising: identifying a human subject with Alzheimer's disease in need of such treatment by determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers in the sample indicates that the subject suffers from Alzheimer's disease, and If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

4. A method of selecting one or more human subjects for treating Alzheimer's disease or alleviating a symptom thereof, the method comprising: determining the level of one or more biomarkers in samples collected from one or more human subjects; wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, If the one or more biomarkers are detected in the sample, selecting the subject for treatment, and Optionally, an Alzheimer's disease treatment is administered.

5. A method for diagnosing and treating a subject at risk of developing Alzheimer's disease, the method comprising, determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers in the sample indicates that the subject is at risk for developing Alzheimer's disease, and If the one or more biomarkers are detected, an Alzheimer's disease treatment is administered.

6. A method for diagnosing Alzheimer's disease in a subject, the method comprising: determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, If the one or more biomarkers are detected, Alzheimer's disease is diagnosed in the subject.

7. A method for determining whether a subject is at risk of developing Alzheimer's disease, the method comprising: determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein detection of the one or more biomarkers indicates that the subject is at risk for developing Alzheimer's disease.

8. A method for diagnosing and treating Alzheimer's disease or alleviating the symptoms thereof in a human subject, the method comprising: determining an increased level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, wherein the increased levels are measured compared to the levels of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease, and If the level of the one or more biomarkers in the sample is increased, an Alzheimer's disease treatment is administered.

9. A method of selecting and treating a subject suffering from Alzheimer's disease, the method comprising: selecting a human subject having Alzheimer's disease by determining an increased level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, and If the level of the one or more biomarkers in the sample is increased, administering an Alzheimer's disease treatment, wherein the increased levels are measured compared to the levels of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease.

10. A method of identifying and treating a subject suffering from Alzheimer's disease, the method comprising: identifying a human subject having Alzheimer's disease by determining an increased level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, If the level of the one or more biomarkers in the sample is increased, administering an Alzheimer's disease treatment, wherein the increase is measured as compared to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual without Alzheimer's disease.

11. A method for selecting a subject in need of treatment for Alzheimer's disease, the method comprising: determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, comparing the level of one or more biomarkers in the sample to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in individuals without Alzheimer's disease, selecting the subject if the level of the one or more biomarkers in the sample is increased compared to the level in an individual without Alzheimer's disease, and Optionally, an Alzheimer's disease treatment is administered to the subject.

12. A method of treating a subject at risk for developing Alzheimer's disease, the method comprising, determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, comparing the level of the one or more biomarkers to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual not at risk for developing Alzheimer's disease, and If the one or more biomarkers are increased in the subject relative to individuals not at risk for developing Alzheimer's disease, an Alzheimer's disease treatment is administered.

13. A method of measuring the effectiveness of a treatment for Alzheimer's disease, the method comprising: To identify subjects with a CDR score greater than 0 or an MMSE score less than 27, determining pre-treatment levels of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, administering Alzheimer's disease treatment, and determining the post-treatment level of one or more biomarkers selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase in a second sample collected from the subject, Wherein a decrease in the post-treatment level of the biomarker in the second sample relative to the first sample indicates effectiveness of the treatment.

14. A method for diagnosing Alzheimer's disease, the method comprising: determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, comparing the level of the one or more biomarkers in the sample to the level of phosphorylated FYN416 and / or phosphorylated p38 MAP kinase in an individual without Alzheimer's disease, and If the one or more biomarkers are increased in the subject relative to individuals without Alzheimer's disease, Alzheimer's disease is diagnosed in the subject.

15. A method for determining whether a subject is at risk of developing Alzheimer's disease, the method comprising: determining the level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are selected from phosphorylated FYN 416 and phosphorylated p38 MAP kinase, comparing the level of the one or more biomarkers in the sample to the level of phosphorylated FYN 416 and / or phosphorylated p38 MAP kinase in an individual not at risk for developing Alzheimer's disease, and If the one or more biomarkers are increased in the sample relative to individuals not at risk for developing Alzheimer's disease, the subject is determined to be at risk for developing Alzheimer's disease.

16. The method of any one of claims 1-15, wherein the one or more biomarkers further comprise phosphorylated Tau protein and / or β-amyloid protein. 17 . The method according to claim 16 , wherein the phosphorylated Tau protein is phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231 and / or phosphorylated Tau 235.

18. The method of claim 17, wherein the β-amyloid proteins are β-amyloid 40 and β-amyloid 42, and wherein the ratio of β-amyloid 42 to β-amyloid 40 is increased relative to a subject having a CDR of 0 or an MMSE score of about 27-30.

19. The method of any one of claims 8-11 or 14, wherein the individual without Alzheimer's disease has a CDR of 0 or an MMSE score of about 27-30.

20. The method of any one of claims 1-19, wherein the level of the one or more biomarkers is determined using antibodies specific for the one or more biomarkers.

21. The method of claim 20, wherein the level of one or more biomarkers is determined by immunohistochemical analysis, Western blot, ELISA, immunoprecipitation and / or flow cytometric analysis.

22. The method of claim 20, further comprising detecting the level of one or more biomarkers using quantitative immuno-PCR, comprising: contacting the sample with a ligand linked to an oligonucleotide specific for the one or more biomarkers, wherein the oligonucleotide comprises a primer sequence unique to the biomarker, allowing the ligand to bind to the biomarker in the sample to form a complex comprising the ligand, the biomarker and the oligonucleotide, Isolation of the complex, and Amplification oligonucleotides.

23. The method of claim 22, wherein the ligand and oligonucleotide are non-covalently linked via a linker.

24. The method of claim 23, wherein the ligand is an antibody, the oligonucleotide is biotinylated, and wherein the linker comprises a streptavidin-protein A chimera.

25. The method of claim 22, wherein the oligonucleotide is amplified by RT-PCR.

26. The method of claim 22, wherein the oligonucleotides are amplified by digital droplet PCR.

27. The method of claim 22, wherein the oligonucleotide further comprises a unique barcode identifying the oligonucleotide, and wherein the oligonucleotide is sequenced to identify the attached ligand.

28. The method of any one of claims 1-16, wherein the levels of at least two of the following are detected: phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid.

29. The method of any one of claims 1-5 and 8-13, wherein the treatment comprises administering to the subject one or more of: (Galantamine), (Rivastigmine), (donepezil), (memantine), or a pharmaceutically acceptable salt or ester thereof.

30. The method of any one of claims 1-5 and 8-13, wherein the treatment comprises administering to the subject Aducanumab TM ).

31. The method of any one of claims 1-30, wherein the sample is blood, serum, tissue, plasma, or cerebrospinal fluid.

32. A method for diagnosing and treating Alzheimer's disease or alleviating the symptoms thereof in a human subject, the method comprising: determining an increased level of one or more biomarkers in a sample collected from the subject, wherein the one or more biomarkers are phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235 and B-amyloid protein, wherein the increased levels are measured as compared to the levels of phosphorylated FYN 416, phosphorylated p38 MAP kinase, phosphorylated Tau 181, phosphorylated Tau 217, phosphorylated Tau 231, phosphorylated Tau 235, and B-amyloid in individuals without Alzheimer's disease, and If the level of the one or more biomarkers in the sample is elevated, an Alzheimer's disease treatment is optionally administered.

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