Method for Detecting b-Isox Precipitates or Captured Proteins as Biomarkers in Biological Fluids

Abstract

This specification describes a method for detecting conformational diseases, aging, and proteinopathies by measuring the presence of b-isox precipitates and the levels of b-isox capture proteins in the biological fluids of healthy individuals and patients. Additional biomarkers have been identified by research, enabling the detection, diagnosis, or treatment of human diseases in human subjects by detecting the biomarkers with or without the addition of isoxazole to the obtained biological fluid samples. It has become possible to use b-iso and / or biomarkers for the diagnosis of diseases.

Description

Technical Field

[0001] Cross-reference This application claims the priority of U.S. Provisional Application No. 63 / 351,813, filed on June 13, 2022, and the entire content of that application is incorporated herein by reference.

[0002] A method of adding isoxazole to a biological fluid sample obtained to form a biological fluid isoxazole composition in the biological fluid sample and detecting the presence of the biological fluid isoxazole composition enables the detection, diagnosis, or treatment of human diseases in a human subject. Additional biomarkers have been identified by research, and by detecting the biomarkers, with or without adding isoxazole to the obtained biological fluid sample, it has become possible to detect, diagnose, or treat human diseases in a human subject.

[0003] This method can also be used for other purposes, and relates to methods for differential diagnosis of conformational diseases and proteinopathies, such as neurodegenerative diseases, diabetes, cancer, mental disorders, and even for differential diagnosis of aging, real-time pathophysiological monitoring, pre-symptomatic diagnosis, and measurement of pharmacological responses.

[0004] Background of the Invention Conformational diseases include more than 50 disorders caused by the accumulation of unfolded or misfolded proteins. Inappropriate protein folding can lead to the deposition of amorphous aggregates such as p-TDP-43 aggregates, or regular amyloid fibers such as synuclein and tau inclusions (1).

[0005] Proteinopathy refers to a pathological condition caused by certain misfolded proteins with an aggregation tendency, such as synuclein, TDP-43, and tau. For example, there are synucleinopathies and tauopathies.

[0006] Amyotrophic lateral sclerosis (ALS) is a progressive heterogeneous neuromuscular disease with limited treatment options (2). The degenerative symptoms of ALS are characterized by the progressive loss of mainly motor neurons in the cerebral cortex, brainstem, and spinal cord in adulthood, resulting in progressive muscle weakness and atrophy, typically leading to death within 3 to 5 years after symptom onset. Several ALS-causing genes, including SOD1, TDP-43, and C9orf72, have been discovered for ALS, and these proteins change into misfolded structures and form pathological inclusions. However, due to the lack of a definitive target, current treatments or diagnoses are still limited (3-7). Currently, the typical time until ALS is diagnosed is 10 to 16 months after symptom onset. Misdiagnosis is a major factor leading to a delay in diagnosis. So far, there are no biomarkers available for sporadic ALS, which accounts for 90% of ALS patients.

[0007] Alzheimer's disease (AD) is the most common cause of memory loss and cognitive impairment (such as dementia) and is thought to account for 60-70% of cases worldwide. The AD brain is characterized by extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary (tau) tangles (8). Both abnormal structures are highly insoluble aggregated amyloid fibrils. Aβ plaques are derived from the proteolytic processing of the transmembrane protein amyloid precursor protein (APP). APP is cleaved by β- and γ-secretases to generate Aβ peptides. There is evidence that excessive Aβ production induces neurotoxicity and triggers the formation of neuronal tangles and neuronal loss in brain regions where plaques have deposited. This process is thought to be the pathogenesis mechanism in both familial and sporadic AD. The second risk factor for AD is tau, a microtubule-associated protein. Tau stabilizes the microtubule network and regulates axonal integrity and axonal transport. Microtubule disruption may be caused by loss of function of tau due to abnormal hyperphosphorylation. Hyperphosphorylated tau isoforms are observed in the AD brain as the main components of neurofibrillary tangles (NFTs) and neuropil threads. At least 19 amino acids are phosphorylated and correlate with the severity of AD. Therefore, the large amount of phosphorylated tau (phosphor-Tau) (pTau-217) isoforms observed in cerebrospinal fluid (CSF) and plasma are used as early diagnostic markers for AD.

[0008] Parkinson's disease (PD) is a progressive neurodegenerative disorder that mainly affects the motor system, usually causing rigidity, bradykinesia, and tremors. As the disease worsens, non-motor symptoms, including cognitive changes, also appear. PD is mainly caused by the loss of dopaminergic neurons in the substantia nigra (SN), a basal ganglia structure located in the midbrain. The characteristic pathology of PD is the Lewy body (LB) (9). Misfolded α-synuclein (α-Syn) is observed as the main component of LBs in sporadic PD, and mutations in α-Syn are associated with several rare familial PD. The second risk factor for PD is leucine-rich repeat kinase 2 (LRRK2). Mutations in the LRRK2 gene are responsible for 5% of familial PD and 3% of sporadic cases. LRRK2 with the G2019 mutation is more likely to form α-Syn inclusions, and an association between LRRK2 and protein misfolding pathology has been established. Currently, it often takes more than a year to complete the diagnostic process, and methods available for early diagnosis and monitoring of disease progression are not currently approved.

[0009] b-isox, biotinylated isoxazole (6-(5-(thiophen-2-yl)isoxazole-3-carboxamide)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanoate), is a small molecule known to precipitate RNA-binding proteins concentrated in stress granules and RNA granules. Many of the proteins precipitated by b-isox contained low complexity (LC) domains involved in interaction with b-isox. LC domains have an inherent irregular structure and self-interact. This new type of self-interacting domain transiently forms a cross-β polymer condensate phase and executes important biological processes, including DNA transcription and replication, chromatin remodeling, nuclear pore passage, signal transduction, synaptic transmission, and cytoskeleton regulation, through homotypic or heterotypic cross-β multimer interactions. In contrast to the in-register stacking of pathological cross-β aggregates, physiological cross-β multimers are loose and reversible (10, 11, 12, 13).

[0010] Currently, cost-effective biomarker tests and non-invasive diagnostic techniques for detecting pathophysiology at the clinical and pre-onset stages of conformational diseases and proteinopathies, particularly neurodegenerative diseases, are unmet medical needs. The present invention addresses this need and other needs.

[0011] Summary of the Invention A method for advancing differential diagnosis, real-time monitoring of pathophysiology, pharmacological response analysis, pre-onset diagnosis, and subtyping of neurodegenerative diseases by detecting the presence of b-isox precipitates or the levels of b-isox capture proteins from a patient's plasma and CSF. Examples of neurodegenerative diseases include, but are not limited to, ALS, AD, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Parkinson's disease without dementia (PDnD), multiple system atrophy (MSA), spinal muscular atrophy (SMA), limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC), and frontotemporal dementia (FTLD).

[0012] Examples of b-isox precipitating proteins for ALS diagnosis include, but are not limited to, p-TDP-43, SOD1, GR repeat protein (poly(GR)), GA repeat protein (poly(GA)), GP repeat protein (poly(GP)), carbonic anhydrase 1 (CA1), cluster of differentiation 14 (CD14), myosin light chain 12B (MYL12B), peroxiredoxin 2 (PRDX2), stomatin (STOM), profilin 1 (PFN1), β-actin (ACTB), glucose transporter 1 (GLUT-1), survival of motor neuron 1 (SMN), and annexin A5 (ANXA5).

[0013] Examples of b-isox precipitated proteins for PD diagnosis include, but are not limited to, p-TDP-43, CA1, CD14, PRDX2, STOM, ANXA5, synuclein, cell adhesion molecule L1-like (CHL1), RUVB-like AAA ATPase 1 (RUVBL1), neuronal EGFL-like 2 (NELL2), ankyrin-1 (ANK1), and neuronal cell adhesion molecule (NrCAM).

[0014] Examples of b-isox precipitated proteins for AD diagnosis include, but are not limited to, amyloid β, phospho-TDP-43, TDP-43, tau, STOM, and ANK1.

[0015] A method for advancing the pre-symptomatic diagnosis of SOD1 hereditary and sporadic ALS by detecting the levels of b-isox capture GR repeat proteins from the plasma and CSF of a patient.

[0016] A method for detecting a human disease in a subject includes detecting the presence of a biofluid isoxazole precipitate in a biofluid sample of the subject. In some embodiments, in the method of claim 1, the isoxazole in the isoxazole precipitate is biotin-isoxazole, (6-(5-(thiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), or a salt or analog thereof.

[0017] In some embodiments, the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), or frontotemporal lobar dementia (FTLD).

[0018] In some embodiments, the biological fluid is cerebrospinal fluid (CSF) or plasma.

[0019] A method of detecting a human conformational disease in a subject includes detecting the presence of a biological fluid isoxazole capture protein in a biological fluid sample of the subject.

[0020] In some embodiments, the isoxazole is biotin-isoxazole (6-(5-(thiophen-2-yl)isoxazole-3-carboxamide)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate) or an analog thereof.

[0021] In some embodiments, the human conformational disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), or frontotemporal dementia (FTLD), or diabetes.

[0022] In some embodiments, the isoxazole capture protein is detected by a polypeptide.

[0023] In some embodiments, the polypeptide is (a) an antibody, or (b) an immunoglobulin chain or its binding domain that binds to an isoxazole capture protein. In some embodiments, the human conformational disease is ALS, and the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phospho-TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB or GLUT1. In some embodiments, the human conformational disease is AD, and the polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, tau, STOM or ANK1. In some embodiments, the human conformational disease is PD, and the polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 and RUVBL1. In some embodiments, the biological fluid is plasma and CSF.

[0024] A method for detecting a conformational disease in a subject includes detecting the biological fluid level of a protein having a cross-β structure in a sample of the subject.

[0025] In some embodiments, the biological fluid is plasma and CSF. In some embodiments, the protein having a cross-β structure is detected by a combination of a cross-β recognition probe and a polypeptide. In some embodiments, the conformational disease is ALS, AD, PDD, PDnD, DLB, MSA, SMA, FTLD, diabetes, schizophrenia, cancer, aging, and limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC), or diabetes.

[0026] Use of a reagent for detecting a b-isox capture complex in the manufacture of a kit for assessing the risk of onset or progression of a conformational disease.

[0027] A method for detecting aging and conformational diseases in a subject includes detecting the presence of low-complexity protein complexes in a sample of the subject.

[0028] A method for detecting sporadic ALS in a subject includes detecting the presence of dipeptide repeat proteins in a sample of the subject.

[0029] In some embodiments, the dipeptide repeat proteins are poly(GR), poly(GP), and poly(GA).

[0030] A method for detecting sporadic ALS and SOD1 hereditary ALS in a subject at the prodromal and pre-symptomatic stages includes detecting the presence of dipeptide repeat proteins in a sample of the subject.

[0031] In some embodiments, the dipeptide repeat protein is poly(GR).

[0032] As used in this patent, the terms "invention", "the invention", "this invention", and "the present invention" are intended to broadly refer to the subject matter of this patent and all of the following claims. Statements containing these terms are not to be construed as limiting the subject matter described herein or the meaning or scope of the following claims. Embodiments of the invention that are the subject of this patent are defined by the following claims, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the detailed description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any or all of the drawings, and the appropriate portions of each claim.

[0033] The present invention will become more apparent when read in conjunction with the accompanying drawings and the following detailed description.

[0034] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed invention.

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (some) embodiments of the invention and, together with the specification, serve to explain the principles of the invention.

[0036] Exemplary embodiments of the present invention will be described in detail below with reference to the following drawings:

Brief Description of the Drawings

[0037]

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[0038] Description of Embodiments Here, the present embodiments (exemplary embodiments) of the present invention are referred to in detail, and examples thereof are shown in the accompanying drawings. As far as possible, the same reference numerals are used throughout the drawings to refer to the same or similar parts.

[0039] Provided herein is a method for detecting conformational diseases and proteinopathies, such as neurodegenerative diseases, by using small molecules to generate visible precipitates.

[0040] In addition, the applicants have identified novel specific biomarker in biofluids for differential diagnosis and monitoring of pathophysiology of patients with neurodegenerative diseases, including ALS, AD, and PD.

[0041] Also described is a novel method for plasma diagnosis of neurodegenerative diseases. This novel method, named b-isox-ELISA, combines b-isox chemical precipitation with an immunoassay using a biomarker specific for ALS, AD, or PD. B-isox ELISA can be used for screening the risk of ALS, AD, and PD. The present invention can be used for the discrimination of AD, TDP-43 proteinopathy, PD, and Lewy body dementia, ALS subtyping, real-time readout of pharmacological response, and monitoring of reduction of pathological burden of misfolding disease proteins in clinical trials, preclinical diagnosis, and clinical practice.

[0042] Definitions As used above and throughout this disclosure, the following terms are to be understood to have the following meanings unless otherwise indicated.

[0043] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0044] Exemplary Embodiments Embodiment 1. A method for detecting a human disease in a human subject, comprising, optionally, obtaining a biofluid sample from the subject, adding isoxazole to the obtained biofluid sample to form a biofluid isoxazole composition in the biofluid sample, and detecting the presence of the biofluid isoxazole composition.

[0045] Embodiment 2. The method according to Embodiment 1, wherein the isoxazole is biotin-isoxazole, (6-(5-(thiophen-2-yl)isoxazole-3-carboxamide)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta noate), or a salt or analog thereof, particularly biotin-isoxazole or a salt thereof, and most particularly biotin-isoxazole.

[0046] Embodiment 3. The method according to Embodiment 1 or 2, wherein the biological fluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as urine, saliva, CSF, or plasma, particularly CSF or plasma. In some embodiments, the biological fluid sample is in the form of a body fluid such as urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, and optionally, the biological fluid sample is further processed, for example, by a technique for removing some components, such as concentrating components such as proteins by chemical precipitation. In some embodiments, the biological fluid sample is blood, plasma, or serum, CSF, urine, or saliva. In some embodiments, the biological fluid sample is plasma or CSF.

[0047] Embodiment 4. The human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious disease, Huntington's disease, schizophrenia, age-related disease, or proteinopathy, and in particular, the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD), the method according to any one of Embodiments 1 to 3.

[0048] Embodiment 5. The method according to any one of Embodiments 1 to 4, further comprising treating the human disease or, subsequently, modifying an existing treatment, such as a treatment including drug therapy for the human disease, based on the detection of the presence of the biological fluid isoxazole composition. In some embodiments, the method further comprises diagnosing the human disease.

[0049] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the concentration of isoxazole ranges from 0.075 mM to 0.225 mM, preferably from 0.100 mM to 0.200 mM, in the biological fluid sample.

[0050] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the biological fluid isoxazole composition is in a precipitate.

[0051] Embodiment 8. The method according to Embodiment 7, wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

[0052] Embodiment 9. The method according to Embodiment 8, wherein the human disease is ALS, AD, PDD, DLB, MSA or PDnD.

[0053] Embodiment 10. The method according to Embodiment 9, wherein the human disease is ALS such as sporadic ALS.

[0054] Embodiment 11. The method according to any one of Embodiments 7 to 10, further comprising monitoring the size of the precipitate to monitor the progression of ALS in a subject.

[0055] Embodiment 12. The method according to any one of Embodiments 1 to 6, further comprising adding a polypeptide to the biological fluid isoxazole composition in a biological fluid sample to facilitate detection by an immunoassay, such as a blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light-scattering immunoassay, radiolabeled immunoassay, particularly ELISA or Western blot.

[0056] Embodiment 13. The method according to Embodiment 12, wherein the human conformational disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

[0057] Embodiment 14. The method according to Embodiment 12 or 13, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain or its binding domain that binds to a biological fluid isoxazole composition.

[0058] Embodiment 15. The human disease is ALS, and the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phospho-TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1, such as SOD1, MYL12B, CD14, and p-TDP-43, particularly antibodies against SOD1 and p-TDP-43. The method according to Embodiment 14.

[0059] Embodiment 16. The human disease is AD, and the polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, tau, STOM, or ANK1. The method according to Embodiment 14.

[0060] Embodiment 17. The human disease is PD, and the polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1. The method according to Embodiment 14.

[0061] Embodiment 18. The method according to any one of Embodiments 15 to 17, wherein the biological fluid is plasma or CSF.

[0062] Embodiment 19. The method according to any one of Embodiments 12 to 18, which is an ELISA such as direct, indirect, sandwich or competitive ELISA.

[0063] Embodiment 20. Use of isoxazole for detecting human diseases in the method according to any one of Embodiments 1 to 19.

[0064] Embodiment 21. A method for detecting a human disease in a human subject, comprising detecting the presence of a biomarker from a obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject, the human disease being selected from sporadic amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), and Parkinson's disease without dementia (PDnD), for sporadic ALS, the biomarker being selected from C9orf72 dipeptide repeat, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN and GLUT1, for AD, the biomarker being selected from STOM and ANK1, for PDD or PDnD, the biomarker being selected from CHL1, NELL2, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 and RUVBL1.

[0065] Embodiment 22. The method according to Embodiment 21, wherein the obtained biological fluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as urine, saliva, CSF or plasma, particularly CSF or plasma. The biological fluid sample includes those described in Embodiment 3.

[0066] Embodiment 23. The method according to Embodiment 21 or 22, wherein the human disease is sporadic ALS.

[0067] Embodiment 24. The method according to Embodiment 21 or 22, wherein the human disease is AD.

[0068] Embodiment 25. The method according to embodiment 21 or 22, wherein the human disease is DLB, MSA, PDD or PDnD.

[0069] Embodiment 26. The method further comprising subsequently treating the human subject for the human disease or subsequently modifying an existing treatment of the human subject for the human disease, such as a treatment comprising drug therapy for the human disease, based on the detection of the presence of the biofluid isoxazole composition, and optionally further comprising diagnosing the human disease in the human subject, according to any one of embodiments 21 to 25.

[0070] Embodiment 27. The method according to any one of embodiments 21 to 26, further comprising adding a polypeptide to the obtained biofluid sample to facilitate detection by an immunoassay, such as a blot assay, a chemiluminescent immunoassay, an enzyme-linked immunosorbent assay (ELISA), a light-scattering immunoassay, a radiolabeled immunoassay, particularly ELISA or Western blot.

[0071] Embodiment 28. The method according to embodiment 27, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain or its binding domain that binds to the biomarker.

[0072] Embodiment 29. The method according to embodiment 28, wherein the human disease is sporadic ALS and the polypeptide for detecting sporadic ALS is an antibody against C9orf72 dipeptide repeat, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as an antibody against C9orf72 dipeptide repeat.

[0073] Embodiment 30. The method according to embodiment 28, wherein the human disease is AD and the polypeptide for detecting AD is an antibody against STOM or ANK1.

[0074] Embodiment 31. The method according to embodiment 28, wherein the human disease is DLB, MSA, PDD or PDnD, and the polypeptide for detecting PD is an antibody against CHL1, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 or RUVBL1.

[0075] Embodiment 32. The method according to any one of embodiments 29 to 31, wherein the biological fluid is plasma or CSF.

[0076] Embodiment 33. The method according to any one of embodiments 27 to 32, which is ELISA such as direct, indirect, sandwich or competitive ELISA.

[0077] Embodiment 34. Use of a biomarker for detecting a human disease in the method according to any one of embodiments 21 to 33.

[0078] Embodiment 35. A method for detecting sporadic amyotrophic lateral sclerosis (ALS) in a human subject, comprising detecting the presence of a dipeptide repeat protein in a obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject to obtain the obtained biological fluid sample.

[0079] Embodiment 36. The method according to embodiment 35, wherein the biological fluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as urine, saliva, CSF or plasma, particularly CSF or plasma.

[0080] Embodiment 37. The method according to embodiment 35 or 36, wherein the dipeptide repeat protein is selected from poly(GR), poly(GP) and poly(GA).

[0081] Embodiment 38. Subsequently, further comprising treating a human subject for a human disease or, subsequently, modifying an existing treatment of a human subject for a human disease, such as a treatment including drug therapy for a human disease, based on the detection of the presence of the biofluid isoxazole composition, and optionally further comprising diagnosing a human disease in the human subject, the method according to any one of Embodiments 35 to 37.

[0082] Embodiment 39. The method according to any one of Embodiments 35 to 38, further comprising adding a polypeptide to the obtained biofluid sample to facilitate detection by an immunoassay, such as a blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light-scattering immunoassay, radiolabeled immunoassay, particularly ELISA or Western blot.

[0083] Embodiment 40. The method according to Embodiment 39, wherein the polypeptide is (a) an antibody or (b) an immunoglobulin chain or its binding domain that binds to a biomarker.

[0084] Embodiment 41. The method according to Embodiment 40, wherein the human disease is sporadic ALS and the polypeptide for detecting sporadic ALS is a dipeptide repeat protein, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as an antibody against the C9orf72 dipeptide repeat.

[0085] Embodiment 42. The method according to any one of Embodiments 39 to 41, wherein the biofluid is plasma or CSF.

[0086] Embodiment 43. The method according to any one of Embodiments 39 to 42, which is an ELISA such as direct, indirect, sandwich or competitive ELISA.

[0087] Embodiment 44. Use of a dipeptide repeat protein for detecting sporadic ALS in the method according to any one of Embodiments 35 to 43.

[0088] Embodiment 45. A method for detecting sporadic or SOD1 hereditary amyotrophic lateral sclerosis (ALS) in a human subject at the prodromal symptom stage and the prodromal stage, comprising detecting the presence of a dipeptide repeat protein from a obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject and obtaining the obtained biological fluid sample.

[0089] Embodiment 46. The method according to embodiment 45, wherein the biological fluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as urine, saliva, CSF or plasma, particularly CSF or plasma.

[0090] Embodiment 47. The method according to embodiment 45 or 46, wherein the dipeptide repeat protein is poly(GR).

[0091] Embodiment 48. Subsequently, treating the human subject for a human disease, or subsequently changing an existing treatment of the human subject for a human disease, such as a treatment including drug therapy for the human disease, based on the detection of the presence of a biological fluid isoxazole composition, and optionally further including diagnosing the human disease in the human subject. The method according to any one of embodiments 45 to 47.

[0092] Embodiment 49. The method according to any one of embodiments 45 to 48, further comprising adding a polypeptide to the obtained biological fluid sample to facilitate detection by an immunoassay, such as a blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light scattering immunoassay, radiolabeled immunoassay, particularly ELISA or Western blot.

[0093] Embodiment 50. The method according to embodiment 49, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain or its binding domain that binds to a biomarker.

[0094] The method according to embodiment 50, wherein the polypeptide for detecting sporadic or SOD1 hereditary ALS is an antibody against a dipeptide repeat protein, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as a C9orf72 dipeptide repeat.

[0095] The method according to any one of embodiments 49 to 51, wherein the biological fluid is plasma or CSF.

[0096] The method according to any one of embodiments 49 to 52, which is an ELISA such as direct, indirect, sandwich or competitive ELISA.

[0097] Use of a dipeptide repeat protein for detecting sporadic or SOD1 hereditary ALS in the method according to any one of embodiments 45 to 53.

[0098] Use of any of the substances or compositions described for diagnosing human diseases in each of embodiments 1 to 53.

[0099] A method for diagnosing, monitoring and predicting the disease progression of neurodegenerative diseases by detecting b-isox precipitates from a patient's plasma The applicants used the small molecule compound b-isox to generate precipitates that can be visually observed in samples from patients with ALS, AD and PD, but not in samples from healthy individuals (Figure 1). The b-isox precipitates accurately discriminated between healthy individuals and ALS patients (Figure 1A).

[0100] The applicants found that plasma (0.1 mL) was sufficient for this diagnosis (Figure 1C) and that a reaction time of 60 minutes reached the optimal conditions (Figure 1B). The accuracy of this diagnosis for ALS, AD and PD was 98.4%, 81.8% and 92.8% respectively (Figure 1D).

[0101] The size of the b-isox precipitate was negatively correlated with the functional score (ALSFRS-R), suggesting that the b-isox precipitate can be used not only for diagnosis but also for predicting the progression to future ALS impairment (Figure 1E).

[0102] Method for differential diagnosis, pre-symptomatic diagnosis, subtyping, and pharmacological response analysis of ALS by detecting b-isox capture proteins in CSF and plasma The applicants developed a novel chemical ELISA method called b-isox ELISA to detect the levels of specific proteins in the b-isox precipitate (Figure 2a).

[0103] The applicants found that the levels of specific b-isox capture proteins tested in plasma were detected by chemical ELISA and were significantly different between the neurodegenerative disease group and healthy controls (Figures 2, 3, 9, and 10).

[0104] The applicants further found that the percentage of patients with high levels of misfolded proteins in plasma, including SOD1, p-TDP-43, and C9orf72, closely corresponded to the percentage of patients known to have inclusions in the spinal cord from autopsy studies, as shown in Figure 2. From these results, it was suggested that b-isox-based ELISA can detect real-time conformational changes of representative known disease-causing proteins, including TDP-43, SOD1, and C9orf72 dipeptide repeat (poly(GR)) proteins, in the plasma of ALS patients.

[0105] The applicants further identified novel pathophysiological biomarkers of ALS by proteomic analysis of the b-isox precipitate, and further reproduced and verified the identified biomarker candidates in another patient cohort (Figures 3 and 4). The levels of these newly identified biomarkers tested in plasma were significantly different between the disease group and healthy individuals.

[0106] Edaravone, an antioxidant clinically used for ALS, reduces the plasma level of PRDX2 but not the plasma levels of TDP-43 or GR repeat proteins, revealing that the ineffectiveness of edaravone in clinical trials may be due to its inability to remove misfolded proteins and confirming the potential use of b-isox ELISA in the pharmacological response analysis of ALS (Figure 6).

[0107] All of the applicants' data suggested that plasma b-isox capture proteins could function as a sensitivity indicator for pathophysiological changes during the onset of neurodegenerative diseases, enabling a precision medicine approach to neurodegenerative diseases.

[0108] Method for diagnosing PD by detecting b-isox capture proteins in CSF and plasma The applicants developed a new chemical ELISA method called b-isox ELISA to detect the levels of specific proteins in b-isox precipitates (Figure 2a).

[0109] The applicants found that the levels of specific b-isox capture proteins tested in plasma were detected by chemical ELISA and were significantly different between the neurodegenerative disease group and HC (Figures 8 and 9).

[0110] The applicants further identified novel biomarker candidates for PD by proteomic analysis of b-isox precipitates and then reproduced and verified the identified biomarker candidates in another patient cohort (Figures 8 and 9). The levels of these novel biomarkers tested in plasma were significantly different between the disease group and healthy individuals.

[0111] Method for diagnosing AD by detecting b-isox capture proteins in CSF and plasma The applicants developed a new chemical ELISA method called b-isox ELISA to detect the levels of specific proteins in b-isox precipitates (Figure 2a).

[0112] The applicants detected that the levels of specific b-isox capture proteins tested in plasma were significantly different between the neurodegenerative disease group and HC by chemical ELISA (Figure 10).

[0113] The applicants further identified novel biomarker candidates in biological fluids for AD by proteomic analysis of b-isox precipitates, and further reproduced and validated the identified biomarker candidates in another patient cohort (Figure 10). The levels of these novel biomarkers tested in plasma were significantly different between the disease group and healthy individuals.

[0114] Poly(GR) as a novel pre-symptomatic biomarker for hereditary SOD1 and sporadic ALS Aggregate depositions of dipeptide repeat proteins (polyGR, GP or GA) were characteristic of C9orf72 hereditary ALS. Unexpectedly, GR repeat proteins appeared at an early stage, constantly maintained high levels in the plasma of 87% of ALS patients, and were the basis for the disease progression of ALS (Figure 4, and 7a). Since hereditary patients account for only up to 10% of ALS, these results suggest that poly(GR) proteins are increased in the plasma of sporadic ALS. These results shed light on the new pathophysiological role of plasma dipeptide repeat proteins (polyGR, GP or GA).

[0115] An increase in poly(GR) levels is not observed in SMA, AD and PD (Figure 7b).

[0116] The applicants identified that an increase in poly(GR) proteins appears at the pre-symptomatic stage of hereditary individuals with C9orf72 mutations (Figure 7c) and at the pre-onset stage of the SOD1 mouse model (Figure 7d). Therefore, it was suggested by the applicants that poly(GR) functions as a pre-symptomatic biomarker for C9orf72 ALS, sporadic ALS and SOD1 hereditary ALS.

[0117] Furthermore, the applicants also found that plasma poly(GA) and poly(GP) were increased in more than 70% of the patients. The applicants suggested that poly(GA) and poly(GP) could be used as plasma biomarkers for the diagnosis of not only C9orf72 hereditary ALS but also sporadic ALS (Figure 2).

[0118] Description of Materials and Methods Used in the Examples The following materials and methods were used in the following examples.

[0119] Reagents and Antibodies: b-isox was purchased from Sigma and Dalton and dissolved in dimethyl sulfoxide (DMSO). The primary antibody against SMN was purchased from BD Bioscience. The primary antibody against GFP was purchased from Roche. The primary antibodies against CA1 (#MBS1492724) and PRDX2 (#MBS7046127) were purchased from Mybiosource. The primary antibodies against MYL12B (#10324-1-AP), CD14 (#17000-1-AP), α-synuclein (#10842-1-AP), GR repeat protein (#23978-1-AP), phospho-TDP-43 (Ser409 / 410) (#22309-1-AP), PFN1 (#11608-1-AP), NrCAM (#14255-A-AP), STOM (#12046-1-AP), CHL1 (#25250-1-AP), NELL2 (#11268-1-AP), RUVBL1 (#10210-2-AP), ANXA5 (#11060-A-AP) and GLUT-1 (#21829-1-AP) were purchased from Proteintech. The primary antibody against β-actin (#A1978) was purchased from Sigma. The primary antibodies against tau (#T9450) and ANK1 (#PA5-42203) were purchased from ThermoFisher. The primary antibody against SOD1 (#A2770) was purchased from Cell signaling.

[0120] b-isox precipitation: 10 mM biotinylated isoxazole was added to human plasma or CSF to a final concentration of 100 - 200 μM. The mixture was then incubated at 4°C for 60 minutes, centrifuged at 15,000 rpm at 4°C for 15 minutes, and the supernatant was discarded. The diameter of the b-isox precipitate was measured.

[0121] b-isox precipitation and enzyme-linked immunosorbent assay (ELISA): Blood samples from patients and healthy controls were first collected using blood collection tubes. The tubes were centrifuged at 2200 g for 15 minutes. The supernatant (upper layer) was obtained as the plasma sample. Before the immunoassay, 50 - 100 μL of plasma (or CSF) and 0.5 - 1 mL of b-isox were gently mixed by pipetting and rotated at 4°C for 1 hour. Each microplate well coated with streptavidin was washed three times with 200 μL of wash buffer (WB / (25 mM Tris, 150 mM NaCl; pH 7.2), 0.1% BSA, 0.05% Tween®-20) (do not dry the wells). 100 μL of the reaction mixture was added to each well and incubated for 2 hours with shaking (approx. 60 rpm) at room temperature (RT). After the reaction was completed, it was washed three times with 200 μL of WB. 100 μL of the primary antibody diluent (appropriate primary antibody dilution in WB) was added and incubated for 1 hour with shaking at RT. In this step, a control without the primary antibody needs to be added (add only the antibody diluent to the sample wells). It was washed three times with 200 μL of WB. To 100 μL of the antibody diluent, an appropriately diluted HRP-conjugated secondary antibody was added and incubated for 1 hour with shaking at RT. In this step, the TMB substrate solution was equilibrated to RT. It was washed three times with 200 μL of WB. 100 μL of the TMB substrate solution was added to each microplate well and incubated for 15 - 30 minutes until color developed. 100 μL of 2 M sulfuric acid (or 2 N HCl) was added to stop the reaction. The optical density at 450 nm was measured using a microplate reader.

[0122] Removal of misfolded proteins from plasma: Human plasma samples were incubated overnight at 4°C with rotation (30 rpm) with A11 or B8H10 antibody. On the second day, the reaction samples were incubated for 1 hour at room temperature with rotation (30 rpm) with 5 μl of magnetic bead conjugated Protein A / G beads (ThermoFisher). The mixture was then placed on a magnet and separated at RT for 3 minutes into antibody-depleted plasma and antibody-captured proteins. The supernatant was collected and further tested by b-isox ELISA using specific biomarkers.

[0123] Example Example 1: Analysis of the amount of b-isox precipitate from plasma of healthy controls and patients with ALS, AD, and PD One milliliter of plasma from normal subjects and ALS patients was incubated with b-isox and centrifuged to pull down cross-β prion-like LC proteins (Figure 1a). b-isox precipitates can be visually observed in patient samples but not in healthy subject samples (Figure 1a). The optimal condition for b-isox precipitation is to incubate 100 μl of plasma with b-isox for 60 minutes (Figures 1b and c).

[0124] Figure 1d shows the statistical analysis of the size of b-isox precipitates from 100 μl of plasma from normal subjects and patients with ALS, AD, and PD. The accuracy of b-isox precipitation for ALS, AD, and PD is 98.4%, 81.8%, and 92.8%, respectively.

[0125] Follow-up analysis of two ALS patients showed that the size of b-isox precipitates was negatively correlated with the functional score (ALSFRS-R) (Figure 1e). These results suggest that b-isox precipitates can be used not only for diagnosis but also for predicting the progression to future ALS impairment.

[0126] Example 2: Differential diagnosis of ALS by b-isox ELISA To detect the levels of specific low-complexity (LC) proteins in the b-isox precipitate, a chemical ELISA method called b-isox ELISA was developed. The schematic diagram of b-isox ELISA is shown in Figure 2a. After mixing plasma with b-isox to generate b-isox binding complexes, streptavidin was used to capture the b-isox binding complexes, and then conventional ELISA was performed using a specific antibody against the target of interest.

[0127] First, the levels of b-isox-captured SOD1 in the plasma of control, ALS mice with the SOD1 G93A mutation, and SMA mice were analyzed by b-isox-based ELISA (Figure 2b). An increase in the level of SOD1 in the precipitate isolated from the plasma of ALS mice was found to be related to the time when misfolded SOD1 protein aggregates in ALS mice. The level of SOD1 did not change in the plasma of SMA mice.

[0128] Consistently, the levels of SOD1 in the plasma of patients with the hereditary SOD1 G93A mutation were examined, and it was found that in hereditary patients with ALSFRS-R 43, the plasma level of SOD1 was significantly increased compared to the control (Figure 2c).

[0129] Furthermore, the analysis of about 120 ALS patients showed that about 10% of the patients with high plasma SOD1 closely corresponded to the proportion of patients known to have SOD1 inclusions in the spinal cord from autopsy studies, as shown in Figure 2d.

[0130] Next, the levels of p-TDP-43 in the precipitate isolated from the plasma of ALS patients were examined by b-isox ELISA. Consistently, it was found that the positive rate of patients with p-TDP-43 correlated with the reported incidence of TDP-43 in ALS (Figure 2e).

[0131] In patients with high plasma levels of p-TDP-43, the plasma levels of GR repeat proteins were also increased, but the levels of TDP-43 and SOD1 were not increased (Figure 2f).

[0132] Longitudinal studies revealed that both plasma levels of p-TDP-43 and the size of b-isox deposits decreased during the progression of ALS (Figure 2g).

[0133] Next, plasma levels of GR repeat proteins were examined in normal controls and ALS patients. The positive rate of patients with elevated GR repeat proteins was up to approximately 87.5%, covering the sporadic ALS group and not reported previously (Figure 2h).

[0134] In a representative profile of ALS patients, plasma p-TDP-43 and SOD1 proteins were shown not to be elevated even in patients with high plasma levels of GR repeat proteins (Figure 2i).

[0135] Figure 2j shows the longitudinal follow-up of the relationship between plasma levels of GR repeat proteins, the size of b-isox deposits, and ALSFRS-R.

[0136] Example 3: Identification and Validation of Novel Pathophysiological Biomarkers for ALS Using proteomic analysis of b-isox-generated deposits recovered from the CSF of patients with ALS, AD, or PD, several novel potential biomarkers of pathophysiology involved in the pathogenesis of ALS, AD, or PD were discovered. The comprehensive flowchart of the experiment is shown in Figure 3a. b-isox capture deposits from plasma of healthy individuals and patients with ALS, AD, or PD were subjected to protein identification by LC-Ms / Ms, and differentially expressed proteins were classified according to enriched gene ontology (GO) terms into membrane transport, exosomes, metabolism, spliceosome, peptides, and inhibitors, and chromosomes, and related proteins.

[0137] By reproducing and validating the identified biomarker candidates in another patient cohort, CA1, CD14, MYL12B, PRDX2, STOM, GLUT-1, SMN, β-actin (ACTB), PFN1, poly(GA), and poly(GP) were confirmed as biomarkers of pathophysiology in ALS (Figure 3b). The levels of nine b-isox capture proteins tested in plasma were significantly different between the disease group and healthy individuals.

[0138] Unexpectedly, the applicants found that poly(GR), poly(GA), and poly(GP), which are dipeptide repeat proteins in plasma, were increased in more than 80% of patients, suggesting that these proteins can be used as biomarkers not only for C9orf72 hereditary ALS but also for the diagnosis of sporadic ALS (Figures 2 and 3).

[0139] Example 4: Association between Plasma b-isox Capture Proteins and ALS Impairment To explore the relationship between the proteinopathy of modulator / risk proteins and the progression to impairment in ALS patients, the association between the levels of pathogenic variants and the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) scores was analyzed. The plasma levels of pathogenic variants including p-TDP-43, GR repeat protein, MYL12B, STOM, SOD1, CD14, CA1, PRDX2, and ANXA5 were measured. The patients were subgrouped into four stages according to the functional scores (Figure 4a). The ALSFRS-R is divided into four groups: stage I (score 40 - 48), stage II (score 30 - 39), stage III (score 20 - 29), and stage IV (score 10 - 19). The upper percentages represent the proportion of patients in whom the biomarker is increased.

[0140] In addition to high SOD1 being seen in the early stage of ALS, a subgroup with low SOD1 appears in the later stage. Since SOD1 inclusions have been detected in the motor neurons of sALS patients with an increase in C9ORF72 repeats even without SOD1 mutations, it was inferred that the induction of SOD1 misfolding is a common downstream event in ALS progression (Figure 4a).

[0141] Furthermore, PRDX2, MYL12B, and CD14 also showed a similar trend, which decreased after stage II (score 30 - 39) (Figure 4b).

[0142] High levels of GR repeat proteins are maintained during the progression to the disorder (Figures 4a and c).

[0143] Notably, the presence of high levels of ANXA5 protein in the plasma of ALS patients only at the initial stage of ALS reflects the occurrence of neuronal turnover that can induce the onset of the ALS disease (Figure 4d).

[0144] Example 5: Longitudinal tracking of b-isox capture proteins in the plasma of ALS Twenty-three participants were subjected to b-isox ELISA analysis for the progression to ALS and were followed up while repeating clinical examinations up to 3.5 years. It was observed that the b-isox capture proteins in the plasma of the patients changed dynamically through the disease progression of ALS in individual patients. Two examples are shown in Figure 5.

[0145] Example 6: Pharmacological response analysis of edaravone in ALS By monitoring the real-time pharmacological responses of two ALS patients treated with edaravone using b-isox ELISA, it was found that edaravone treatment decreased plasma PRDX2 and CD14, but had no significant effect on plasma GR repeat proteins (Figure 6). Edaravone is an antioxidant that can explain the decrease in PRDX2. It was inferred that edaravone was ineffective in ALS clinical trials because it could not reduce the pathological burden of misfolded GR repeat proteins.

[0146] Example 7: Poly(GR) is a very early biomarker for SOD1 hereditary and sporadic ALS Among all the ALS biomarkers obtained from the b-isox precipitation analysis, only the GR repeat protein appeared at the early stage, maintained a consistently high level in the plasma of patients, and was the basis for the disease progression of ALS (Figures 4 and 7a). However, an increase in poly(GR) protein was not observed in SMA, AD, and PD, suggesting that the GR repeat protein is a specific biomarker for ALS (Figure 7b).

[0147] A female with hereditary C9orf72 repeats at the pre-symptomatic stage showed an increase in poly(GA), and CD14 and poly(GR) proteins, but no changes in p-TDP-43 and SOD1 (Figure 7c).

[0148] Indeed, in SOD1 G93A mice, it was observed that the plasma GR repeat protein increased at the pre-symptomatic stage and appeared earlier than the plasma misfolded SOD1 (Figure 7d). From these results, it is suggested that the GR repeat protein not only functions as a pre-symptomatic biomarker for hereditary C9orf72 ALS, but can also be used as a very early biomarker involved in sporadic ALS and other possible types of hereditary ALS, such as SOD1 and TDP-43 mutations.

[0149] Example 8: Identification and Validation of Plasma Biomarkers for PD by b-isox ELISA Analysis of PD patients by b-isox ELISA showed that there were significant quantitative differences in the chemical precipitates between PD patients and healthy individuals. Proteomic analysis was used to comprehensively analyze the precipitates isolated from the CSF of PD patients, and several PD biomarker candidates were obtained.

[0150] As expected, it was confirmed by b-isox-ELISA that the levels of synuclein, p-TDP-43, PRDX2, CHL1, NrCAM, STOM, CA1, CD14, ANXA5, NELL2, and RUVBL1 were elevated in the plasma of PD patients (Figure 8).

[0151] Example 9: Molecular profiling of PD, PDD, MSA, and DLB by b-isox ELISA for PD Plasma from patients with PD, PDD, MSA, and DLB was characterized by PD biomarkers (Figure 9).

[0152] Example 10: Identification and validation of plasma biomarkers for AD by b-isox ELISA Analysis of AD patients by b-isox ELISA showed a significant quantitative difference in chemical precipitates between AD patients and healthy individuals. Using proteomic analysis, the precipitates isolated from the CSF of AD patients were comprehensively analyzed.

[0153] As expected, it was confirmed by b-isox-ELISA that the levels of amyloid β, tau, p-TDP-43, TDP-43, ANK1, and STOM were elevated in the plasma of AD patients (Figure 10a).

[0154] According to b-isox-ELISA, ANK1 and STOM showed significantly higher sensitivity than amyloid β (Figure 10b).

[0155] References 1. Prof et al., Conformational disease, 1997, The Lancet, vol. 350, pp.134-138. 2. Kiernan et al., Amyotrophic lateral sclerosis, 2017, The Lancet. Vol. 390, pp. 2084-2098. 3. Rosen et al., Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, 1993, Nature. Vol. 362(6415), pp. 59-62. 4. Neumann et al., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, 2006, Science, vol. 314, pp.130-133. 5. Wang et al., TDP-43: an emerging new player in neurodegenerative diseases, 2008, Trends Mol. Med., vol. 14, pp.479-485. 6. DeJesus-Hernandez et al., Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS, 2011, Neuron. vol.72, pp.245-56. 7. Achmitz et al., Emerging Perspectives on Dipeptide Repeat Proteins in C9ORF72 ALS / FTD, 2021, The Lancet, vol. 397, pp. 1577-1590. 8. Scheltens et al., Alzheimer’s disease, 2021, The Lancet, vol. 397, pp.1577-1590. 9. Braak et al., Staging of brain pathology related to sporadic Parkinson’s disease, 2003, Neurobiol. Aging, vol. 24(2), pp.197-211. 10. Wang et al. The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies, 2012, Nature Commun., Vol. 3, pp. 766. 11. Kato et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels, 2012, Cell, vol. 149, pp. 753-767. 12. Han et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies, 2012, Cell, vol. 149, pp. 768-779. 13. Brangwynne et al. Germline P granules are liquid droplets that localize by controlled dissolution / condensation, 2009, Science vol. 324, pp. 1729-1732.

[0156] Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. This specification and examples are intended to be considered as exemplary only.

Claims

1. A method for detecting a human disease, Adding isoxazole to a biofluid sample taken from a human to form a biofluid isoxazole composition in the biofluid sample, To detect the presence of the aforementioned biological fluid isoxazole composition. Methods that include...

2. The method according to claim 1, wherein the isoxazole is biotin-isoxazole, (6-(5-(thiophen-2-yl)isoxazole-3-carboxamide)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanoate), or a salt thereof or an analog thereof.

3. The method according to claim 1, wherein the biological fluid sample is urine, whole blood, plasma, serum, cerebrospinal fluid (CSF), saliva, or mucous membrane.

4. The method according to claim 1, wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-dominant senile TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes mellitus, cancer, infectious disease, Huntington's disease, schizophrenia, age-related disease, or proteinopathy.

5. The method according to claim 1, wherein the concentration of isoxazole in the biological fluid sample is in the range of 0.075 mM to 0.225 mM.

6. The method according to claim 1, wherein the biofluid isoxazole composition is present in the precipitate.

7. The method according to claim 1, wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic-dominant senile TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

8. The method according to claim 1, wherein the human disease is ALS, AD, DLB, MSA, PDD, or PDnD.

9. The method according to claim 1, wherein the aforementioned human disease is sporadic ALS.

10. The method according to claim 6, further comprising monitoring the size of the precipitate in order to monitor the progression of ALS in a human subject.

11. The method according to claim 1, further comprising adding a polypeptide to the biofluid isoxazole composition in the biofluid sample to facilitate detection by immunoassay, blot assay, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), light scattering immunoassay, radiolabeled immunoassay, or Western blotting.

12. The method according to claim 11, wherein the human disease is a human conformational disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease without dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), limbic-dominant senile TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), and frontotemporal dementia (FTLD).

13. The method according to claim 11, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds to the biofluid isoxazole composition.

14. The method according to claim 13, wherein the human disease is ALS, and the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phospho-TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1.

15. The method according to claim 13, wherein the human disease is AD, and the polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, tau, STOM, or ANK1.

16. The method according to claim 13, wherein the human disease is PD, and the polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1.

17. The method according to claim 11, wherein the biological fluid sample is plasma or cerebrospinal fluid (CSF).

18. The method according to claim 11, wherein it is ELISA.

19. The use of isoxazole for detecting human diseases in the method described in claim 1.

20. The method according to claim 2, wherein the isoxazole is biotin-isoxazole.

21. The method according to claim 3, wherein the biological fluid sample is urine, saliva, cerebrospinal fluid (CSF), or plasma.

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