Method for detecting B-ISOX precipitates or capture proteins as biomarkers for biological fluids

Abstract

Described herein are methods for detecting conformational diseases, aging, and proteinopathies by measuring the presence of b-isox precipitates and the level of b-isox capture proteins in biological fluids of healthy individuals and patients. The studies have identified additional biomarkers that make it possible to detect biomarkers by adding or not adding isoxazole to the obtained biological fluid sample, thereby making it possible to detect, diagnose or treat human diseases in a human subject. Diagnosis of disease using b-isox and / or biomarkers becomes possible.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 351,813, filed on June 13, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] A method for detecting and diagnosing or treating a human disease in a human subject by adding an isoxazole to a obtained biological fluid sample to form a biological fluid isoxazole composition in the biological fluid sample and detecting the presence of the biological fluid isoxazole composition. The study identified additional biomarkers that make it possible to detect, diagnose or treat a human disease in a human subject by detecting the biomarker with or without the addition of an isoxazole to the obtained biological fluid sample.

[0004] Although useful for other purposes, the methods relate to methods for differential diagnosis, real-time pathophysiology monitoring, presymptomatic diagnosis, and drug response measurement of conformational and proteinopathies, such as neurodegenerative diseases, diabetes, cancer, psychiatric disorders, and even aging. Background Art

[0005] Conformational diseases include more than 50 disorders caused by the accumulation of unfolded or misfolded proteins. Improper protein folding can lead to the deposition of amorphous aggregates, such as p-TDP-43 aggregates, or ordered amyloid fibrils, such as synuclein and tau inclusions (1).

[0006] Proteinopathies are pathologies caused by certain misfolded, aggregation-prone proteins, such as synuclein, TDP-43, and tau. Examples include synucleinopathies and tauopathies.

[0007] 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 primary motor neurons in the cerebral cortex, brainstem, and spinal cord with adult onset, leading to progressive muscle weakness, atrophy, and typical death 3 to 5 years after symptom onset. Although a few ALS causative genes have been discovered for ALS, including SOD1, TDP-43, and C9orf72, and their proteins are converted into misfolded structures and form pathological inclusion bodies, current treatment or diagnosis is still hampered by the lack of defined targets (3-7). Currently, the typical time for ALS diagnosis is 10-16 months after symptom onset. Misdiagnosis is a key factor leading to delayed diagnosis. To date, there are no biomarkers for sporadic ALS, which covers 90% of ALS patients.

[0008] Alzheimer's disease (AD) is the most common cause of memory loss and cognitive impairment (e.g., dementia) and may account for 60-70% of cases worldwide. AD brains are characterized by extracellular amyloid-β (Aβ) plaques and intraneuronal neurofibrillary (Tau) tangles (8). Both abnormal structures are highly insoluble and accumulated amyloid fibrils. Aβ plaques are derived from proteolytic processing of the transmembrane protein, amyloid precursor protein (APP). APP is cleaved by β- and γ-secretases to produce Aβ peptides. Evidence suggests that excessive Aβ production induces neurotoxicity, triggering neuronal tangle formation, and neuronal loss in brain regions where plaques are deposited. This process is thought to be the pathogenic mechanism of both familial and sporadic AD. A second risk factor for AD is the microtubule-associated protein Tau. Tau stabilizes the microtubule network and regulates axonal integrity and axonal transport. Disruption of microtubules may result from loss of function due to abnormal hyperphosphorylation of Tau. Hyperphosphorylated Tau isoforms are the main components of neurofibrillary tangles (NFTs) and neuropil threads observed in AD brains. At least 19 amino acids are phosphorylated and correlated with AD severity. Therefore, the high abundance of phospho-Tau (pTau-217) isoform observed in cerebrospinal fluid (CSF) and plasma is used as an early diagnostic marker for AD.

[0009] Parkinson’s disease (PD) is a progressive neurodegenerative disorder that primarily affects the motor system, typically with rigidity, hypokinesia, and tremor. As the disease progresses, non-motor symptoms, including cognitive changes, also develop. PD is primarily caused by the loss of dopaminergic neurons in the substantia nigra (SN), a structure in the basal ganglia located in the midbrain. The hallmark pathology of Parkinson’s disease is Lewy bodies (LBs)(9). Misfolded α-synuclein (α-Syn) is the major component of LBs observed in sporadic Parkinson’s disease, and mutations in α-Syn are associated with some rare forms of common Parkinson’s disease. A second risk factor for Parkinson’s disease is leucine-rich repeat kinase 2 (LARRK2). Mutations in the LARRK2 gene account for 5% of common Parkinson’s disease and 3% of sporadic cases. LRRK2 carrying the G2019 mutation is more susceptible to the formation of α-Syn inclusions, linking LRRK2 to protein misfolding pathology. Currently, the diagnostic process often takes more than a year to complete, and there are currently no recognized methods for early diagnosis and monitoring of disease progression.

[0010] b-ISOX, biotinylated isoxazole (6-(5-(thien-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), is a small molecule known to precipitate RNA-binding proteins enriched in stress granules and RNA granules. Most proteins precipitated by b-ISOX contain a low complexity (LC) domain that is responsible for the interaction with b-ISOX. The LC domain is intrinsically disordered and self-interacting. This novel self-interacting domain transiently forms cross-β-polymeric condensates to execute key biological processes including DNA transcription and replication, chromatin remodeling, nuclear pore pathways, signal transduction, synaptic transmission, and cytoskeletal regulation through homotypic or heterotypic cross-β-polymer interactions. In contrast to the aligned stacking of pathological cross-β aggregates, physiological cross-β aggregates are loose and reversible (10, 11, 12, 13).

[0011] Currently, there is an unmet medical need for cost-effective biomarker tests and non-invasive diagnostic techniques for detecting the clinical and pre-symptomatic pathophysiology of conformational and proteinopathies, particularly neurodegenerative diseases. The present invention addresses this need and others. Summary of the invention

[0012] Methods for advancing the differential diagnosis, real-time monitoring of pathophysiology, analysis of drug response, presymptomatic diagnosis and typing of neurodegenerative diseases by detecting the presence of b-isox precipitates or levels of b-isox-trapped proteins in patient 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 dominant age-related neuropathological changes with TDP-43 encephalopathy (LATE-NC), and frontotemporal dementia (FTLD).

[0013] Examples of b-isox-precipitated 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), stomatoprotein (STOM), profilin 1 (PFN1), β-actin (ACTB), glucose transporter 1 (GLUT-1), survival of motor neuron 1 (SMN), and annexin A5 (ANXA5).

[0014] 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 AAAATPase1 (RUVBL1), neural EGFL-like 2 (NELL2), ankyrin-1 (ANK1), and neuronal cell adhesion molecule (NrCAM).

[0015] Examples of b-isox-precipitated proteins for AD diagnosis include, but are not limited to, amyloid β, phosphorylated TDP-43, TDP-43, Tau, STOM, and ANKL.

[0016] Methods for advancing the presymptomatic diagnosis of SOD1-inherited and sporadic ALS by measuring levels of b-isox-trapped GR repeat proteins in patient plasma and CSF.

[0017] A method for detecting a human disease in a subject comprises detecting the presence of an isoxazole precipitate in a biological fluid sample of the subject. In some embodiments, the method of claim 1, wherein 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 thereof or an analog thereof.

[0018] In some embodiments, the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic dominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathology (LATE-NC) or frontotemporal dementia (FTLD).

[0019] In some embodiments, the biological fluid is cerebrospinal fluid (CSF) or plasma.

[0020] A method of detecting a human conformational disease in a subject comprises detecting the presence of a biological fluid isoxazole trap protein in a biological fluid sample from the subject.

[0021] In some embodiments, the isoxazole 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 an analog thereof.

[0022] In some embodiments, the human conformational disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic dominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathology (LATE-NC) or frontotemporal dementia (FTLD) or diabetes.

[0023] In some embodiments, the isoxazole trap protein is detected by a polypeptide.

[0024] In some embodiments, the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds an isoxazole trap protein. In some embodiments, the human conformational disease is ALS, and wherein the polypeptide used to detect ALS is an antibody to SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phosphorylated TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1. In some embodiments, the human conformational disease is AD, and wherein the polypeptide used to detect AD is an antibody to APP, phosphorylated TDP-43, TDP-43, Tau, STOM, or ANK1. In some embodiments, the human conformational disease is PD, and wherein the polypeptide used to detect PD is an antibody to synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1. In some embodiments, the biological fluid is plasma and CSF.

[0025] A method of detecting a conformational disease in a subject comprises the step of detecting the level of a protein having a cross-beta structure in a biological fluid sample from the subject.

[0026] In some embodiments, the biological fluid is plasma and CSF. In some embodiments, proteins with cross-β structures are detected by a combination of cross-β recognition probes and polypeptides. In some embodiments, the conformational disease is ALS, AD, PDD, PDnD, DLB, MSA, SMA, FTLD, diabetes, schizophrenia, cancer, aging and marginal dominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC) or diabetes.

[0027] The use of a reagent for detecting b-isox capture complexes in the manufacture of a kit to assess the risk of developing a conformational disease or progression of a conformational disease.

[0028] A method for detecting aging and conformational diseases in a subject comprises the step of detecting the presence of low complexity protein complexes in a sample from the subject.

[0029] A method for detecting sporadic ALS in a subject comprises the step of detecting the presence of a dipeptide repeat protein in a sample from the subject.

[0030] In some embodiments, the dipeptide repeat protein is poly(GR), poly(GP), and poly(GA).

[0031] A method for detecting sporadic ALS and SOD1 hereditary ALS in a subject during the presymptomatic and prodromal stages comprises the step of detecting the presence of a dipeptide repeat protein in a sample from the subject.

[0032] In some embodiments, the dipeptide repeat protein is poly(GR).

[0033] The terms "invention," "the invention," "this invention," and "present invention" as used in this patent are intended to refer broadly to all subject matter of this patent and the following patent claims. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the following patent claims. Embodiments of the invention covered by this patent are defined by the following claims rather than by 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 in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all of the drawings, and each claim.

[0034] The present invention will become more apparent upon reading the following drawings and detailed description.

[0035] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0036] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiments of the present invention and, together with the description, serve to explain the principles of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Illustrative embodiments of the invention are described in detail with reference to the following drawings.

[0038] Figure 1is a set of images showing the analysis of b-isox precipitates from plasma of healthy controls and ALS patients. Panel (a) contains a set of images of chemical precipitates isolated from plasma of healthy individuals and ALS patients. Arrows indicate b-isox precipitate particles. At the bottom of each panel is a ruler (mm) used to measure the size of the chemical precipitates. Panel (b) contains a statistical analysis of the time course of chemical precipitates isolated from plasma of healthy individuals and ALS patients. Arrows indicate optimal conditions. Panel (c) contains a statistical analysis of the plasma volume of chemical precipitates isolated from plasma of healthy individuals and ALS patients. Arrows indicate optimal conditions. Panel (d) contains a statistical analysis of the size of b-isox precipitates isolated from 100 μl of plasma of normal, ALS, AD, and PD patients. All data are presented as mean and SD (n for normal, ALS, AD, and PD are 31, 65, 11, and 14, respectively). ****P<0.0001 by Mann-Whitney test. Panel (e) contains a longitudinal follow-up analysis of b-isox precipitates in two ALS patients. The size of b-isox precipitates in the plasma of two ALS patients is measured in months.

[0039] Figure 2is a set of images showing b-isox ELISA and its application in ALS. Figure (a) contains a flow chart of the protocol of b-isox ELISA. Figure (b) contains a set of images showing the level of SOD1 in b-isox precipitates isolated from the plasma of normal controls, SMA mice, and ALS mice by b-isox ELISA. *P<0.05 by Mann-Whitney test. Figure (c) contains a set of images showing the level of b-isox captured SOD1 in the plasma of patients with SOD1 G93A mutation. Figure (d) contains a set of images showing SOD1 typing, b-isox ELISA showing the level of SOD1 in the plasma of normal controls and ALS patients. All data are expressed as mean and SD (n is 38 and 161 for normal and ALS, respectively). ****P<0.0001 by Mann-Whitney test. Figure (e) contains a set of images showing TDP-43 typing, b-isox ELISA showing the level of p-TDP-43 in the plasma of normal controls and ALS patients. All data are presented as mean and SD (n=17 and 115 for normal and ALS, respectively). ****P<0.0001 by Mann-Whitney test. Figure (f) contains a set of images showing biomarker analysis of patients with TDP-43 proteinopathy by b-isox ELISA. Figure (g) contains a set of images showing longitudinal tracking of the association between p-TDP-43 levels, b-isox precipitates, and ALSFRS-R in ALS patients with TDP-43 proteinopathy. Figure (h) contains a set of images showing analysis of plasma GR repeat protein levels in ALS. b-isox ELISA shows the levels of GR repeat protein in plasma of normal controls and ALS patients. All data are presented as mean and SD (n=17 and 111 for normal and ALS, respectively). ****P<0.0001 by Mann-Whitney test. Figure (i) contains a set of images showing biomarker analysis of patients with poly(GR) proteinopathy by b-isox ELISA. Panel (j) contains a set of images showing longitudinal tracking of the association between GR repeat protein levels, b-isox precipitates, and ALSFRS-R in a sporadic ALS patient with poly(GR) proteinopathy.

[0040] Figure 3is a set of images showing the identification and validation of newly discovered ALS biomarkers by b-isox ELISA. Figure (a) contains a set of images showing a schematic diagram of the experimental design for the discovery of new plasma biomarkers for neurodegenerative diseases. Figure (b) contains a set of images showing plasma CA1 levels in healthy individuals (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 20 and 106, respectively). *P<0.05 by Mann-Whitney test. Figure (c) contains a set of images illustrating that b-isox ELISA shows the levels of CD14 in the plasma of healthy controls (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 25 and 95, respectively). *P<0.05 by Mann-Whitney test. Figure (d) contains a set of images illustrating that b-isox ELISA shows plasma MYL12B levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 18 and 91, respectively). *P<0.05 by Mann-Whitney test. Figure (e) contains a set of images illustrating that b-isox ELISA shows plasma PRDX2 levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 22 and 103, respectively). *P<0.05 by Mann-Whitney test. Figure (f) contains a set of images illustrating that b-isox ELISA shows plasma STOM levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 17 and 97, respectively). *P<0.05 by Mann-Whitney test. Figure (g) contains a set of images showing plasma GLUT1 levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n for H and ALS are 12 and 53, respectively). *P<0.05 by Mann-Whitney test. Figure (h) contains a set of images showing plasma SMN levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n is 14 and 58 for H and ALS, respectively). *P<0.05 by Mann-Whitney test. Figure (i) contains a set of images showing plasma β-actin levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n is 8 and 13 for H and ALS, respectively). *P<0.05 by Mann-Whitney test. Figure (j) contains a set of images showing plasma PFN1 levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n is 15 and 43 for H and ALS, respectively). Figure (k) contains a set of images showing plasma poly(GP) levels in healthy controls (H) and ALS patients. All data are expressed as mean and SD (n is 8 and 13 for H and ALS, respectively).*P<0.05 by Mann-Whitney test. Figure (l) contains a set of images showing plasma poly(GA) levels in healthy controls (H) and ALS patients. All data are presented as mean and SD (n for H and ALS are 15 and 43, respectively). *P<0.05 by Mann-Whitney test.

[0041] Figure 4 is a set of images showing the analysis of plasma ALS biomarker levels during the stages of disease progression. Figure (a) contains a set of images showing the levels of 11 biomarkers in plasma, MYL12B, STOM, CA1, p-TDP-43, GR repeat protein, SOD1, CD14, PRDX2, GLUT1, ANXA5, and SMN, measured at various ALS stages. The percentages above represent patients whose levels are above the cutoff values ​​shown by the dotted lines. ALSFRS-R: Stage I (40-48 points), Stage II (30-39 points), Stage III (20-29 points), Stage IV (10-19 points). Figure (b) contains a set of images showing the trend graphs of three biomarkers in ALS progression, including MYL12B, CD14, and PRDX2. Figure (c) contains a set of images showing the changes in CA1, pTDP-43, SOD1, and GR repeat protein in ALS progression. Panel (d) contains a set of images showing changes in ANXA5 during ALS progression.

[0042] Figure 5 is a set of images showing longitudinal tracking of ALS biomarkers. Figures (a) to (c) contain a set of images showing three examples of longitudinal tracking of eight biomarkers, MYL12B, CD14, STOM, PRDX2, p-TDP-43, CA1, SOD1, and GR repeat protein, in ALS patients.

[0043] Figure 6 is a set of images showing analysis of drug response to edaravone in ALS patients by b-isox ELISA. Two examples of pharmacological measurements of edaravone treatment. Arrows indicate treatment with edaravone.

[0044] Figure 7is a set of images showing GR repeat protein as a presymptomatic biomarker for SOD1 inherited and sporadic ALS. Figure (a) contains a set of images showing an overview of combined measurements of plasma biomarkers MYL12B, STOM, CA1, p-TDP-43, GR repeat protein, SOD1, CD14, and PRDX2 in patients with ALSFRS-R scores above 42 by b-isox-based ELISA. Figure (b) contains a set of images illustrating that b-isox ELISA shows plasma GR repeat protein levels in ALS, AD, and PD. All data are presented as mean and SD. Figure (c) contains a set of images illustrating that b-isox ELISA shows plasma SOD1, p-TDP-43, CD14, GR repeat protein, and poly(GA) levels in normal controls and C9orf72 inherited ALS individuals. Figure (d) contains a set of images showing the levels of GR repeat protein in the plasma of normal controls and ALS mice by b-isox-ELISA at the indicated months. All data are presented as mean and SD. *P < 0.05 by Mann-Whitney test.

[0045] Figure 8is a set of images showing the validation of newly identified PD biomarkers by b-isox ELISA. Figure (a) contains a set of images showing plasma synuclein levels in healthy individuals (H) and PD patients. All data are expressed as mean and SD (n=16 and 51 for H and PD, respectively). ****P<0.0001 by Mann-Whitney test. Figure (b) contains a set of images showing that ELISA shows ANXA5 levels in plasma of healthy controls (H) and PD patients. All data are expressed as mean and SD (n=16 and 30 for H and PD, respectively). **P<0.01 by Mann-Whitney test. Figure (c) contains a set of images showing that ELISA shows CA1 levels in plasma of healthy controls (H) and PD patients. All data are expressed as mean and SD (n=20 and 20 for H and PD, respectively). ****P<0.0001 by Mann-Whitney test. Figure (d) contains a set of images showing that ELISA shows plasma CD14 levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n=28 and 20 for H and PD, respectively). ****P<0.0001 by Mann-Whitney test. Figure (e) contains a set of images illustrating that ELISA shows plasma CHL1 levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n=12 and 42 for H and PD, respectively). _***P<0.001 by Mann-Whitney test. Figure (f) contains a set of images illustrating that ELISA shows plasma NrCAM levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n=4 and 34 for H and PD, respectively). ****P<0.0001 by Mann-Whitney test. Figure (g) contains a set of images showing plasma PRDX2 levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n=22 and 13 for H and PD, respectively). ****P<0.0001 by Mann-Whitney test. Figure (h) contains a set of images showing plasma p-TDP-43 levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n for H and PD are 17 and 46, respectively). By Mann-Whitney test, ****P<0.0001. Figure (i) contains a set of images showing plasma RUVBL1 levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n for H and PD are 7 and 13, respectively). By Mann-Whitney test, **P<0.01. Figure (j) contains a set of images showing plasma STOM levels in healthy controls (H) and PD patients. All data are expressed as mean and SD (n for H and PD are 17 and 33, respectively). By Mann-Whitney test, ****P<0.0001.

[0046] Fig. 9 is a set of images showing biomarker analysis of patients with PDnD (a), PDD (b), MSA (c), and DLB (d) by b-isox-ELISA assay.

[0047] Fig.10 is a set of images showing the validation of known and newly identified AD biomarkers by b-isox-ELISA. Figure (a) contains a set of images showing the levels of plasma amyloid β, tau, TDP-43, p-TDP-43, ANK1 and STOM in healthy individuals (H) and AD patients. All data are expressed as mean and SD (n = 12, 4, 2, 17, 14, 2 and 13, 6, 10, 12, 13, 9 for H and AD, respectively). *P<0.05 by Mann-Whitney test. Figure (b) contains a set of images showing the comparison of sensitivity of amyloid β, ANK1 and STOM by b-isox-ELISA. DETAILED DESCRIPTION

[0048] Reference will now be made in detail to the present embodiments of the present invention (exemplary embodiments), examples of which are illustrated in the accompanying drawings. Throughout the drawings, the same reference numerals are used wherever possible to refer to the same or like parts.

[0049] Here we describe methods for detecting conformational and proteinopathies, such as neurodegenerative diseases, by using small molecules to produce visible precipitation.

[0050] Additionally, Applicants have identified novel and specific biofluid biomarkers for differential diagnosis and monitoring pathophysiology in patients with neurodegenerative diseases, including ALS, AD, and PD.

[0051] A novel method for plasma diagnostics of neurodegenerative diseases is also described. This novel method is called b-isox-ELISA, which is a combination of b-isox chemical precipitation and immunoassay with specific biomarkers for ALS, AD or PD. b-isox ELISA can be used to screen for the risk of ALS, AD and PD. The invention can be used to differentiate between AD, TDP-43 proteinopathy, PD and Lewy body dementia, ALS typing, real-time readout of drug response, and monitoring the reduction of the pathological burden of misfolded disease proteins in clinical trials, preclinical diagnostics and clinical practice.

[0052] definition

[0053] As used above and throughout the disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated.

[0054] As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0055] Representative Examples

[0056] Example 1. A method for detecting a human disease in a human subject comprises, optionally obtaining a biological fluid sample from the subject, adding an isoxazole to the obtained biological fluid sample to form a biological fluid isoxazole composition in the biological fluid sample, and detecting the presence of the biological fluid isoxazole composition.

[0057] Embodiment 2. The method of embodiment 1, wherein the isoxazole 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 an analogue thereof, in particular biotin-isoxazole or a salt thereof, very in particular biotin-isoxazole.

[0058] Embodiment 3. The method of embodiment 1 to embodiment 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, especially CSF or plasma. In some embodiments, the biological fluid sample is in the form of a biological 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 chemical precipitation enrichment of components such as proteins to remove some components. 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.

[0059] Example 4. The method of Example 1 to Example 3, wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious disease, Huntington's disease, schizophrenia, aging-related diseases or protein diseases, especially wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy with neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

[0060] Example 5. The method of Example 1 to Example 4, further comprising thereafter treating the human disease or thereafter altering existing treatment based on detecting the presence of the isoxazole composition in the biological fluid, such as treatment including drug therapy for the human disease. In some embodiments, the method further comprises diagnosing the human disease.

[0061] Embodiment 6. The method of embodiments 1 to 5, wherein the concentration of isoxazole in the biological fluid sample ranges from 0.075 mM to 0.225 mM, preferably from 0.100 mM to 0.200 mM.

[0062] Example 7. The method of Examples 1 to 6, wherein the biological fluid isoxazole composition is in a precipitate.

[0063] Example 8. The method of Example 7, wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA) or frontotemporal dementia (FTLD).

[0064] Embodiment 9. The method of embodiment 8, wherein the human disease is ALS, AD, PDD, DLB, MSA or PDnD.

[0065] Embodiment 10. The method of embodiment 9, wherein the human disease is ALS, such as sporadic ALS.

[0066] Example 11. The method of Example 7 to Example 10, further comprising monitoring the size of the precipitate to monitor the progression of ALS in the subject.

[0067] Example 12. The method of Example 1 to Example 6, further comprising adding a polypeptide to the biological fluid isoxazole composition in the biological fluid 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, in particular an ELISA or a Western blot.

[0068] Example 13. The method of Example 12, wherein the human conformational disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA) or frontotemporal dementia (FTLD).

[0069] Embodiment 14. The method of Embodiment 12-13, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds to an isoxazole composition of a biological fluid.

[0070] Example 15. The method of Example 14, wherein the human disease is ALS, and wherein the polypeptide used to detect ALS is an antibody against SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phosphorylated TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB or GLUT1, such as SOD1, MYL12B, CD14 and p-TDP-43, especially SOD1 and p-TDP-43.

[0071] Example 16. The method of Example 14, wherein the human disease is AD, and wherein the polypeptide used to detect AD is an antibody against APP, phosphorylated TDP-43, TDP-43, Tau, STOM or ANK1.

[0072] Example 17. The method of Example 14, wherein the human disease is PD, and wherein the polypeptides used to detect PD are antibodies against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1.

[0073] Embodiment 18. The method of Embodiment 15 to Embodiment 17, wherein the biological fluid is plasma or CSF.

[0074] Embodiment 19. The method of embodiment 12 to embodiment 18, wherein the method is ELISA, such as direct, indirect, sandwich or competitive ELISA.

[0075] Example 20. Using an isoxazole in any of the methods of Examples 1 to 19 to detect a human disease.

[0076] Example 21. A method for detecting a human disease in a human subject, comprising: detecting the presence of a biomarker from an obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject to obtain an obtained biological fluid sample, and wherein the human disease is selected from sporadic amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), and Parkinson's disease non-dementia (PDnD); wherein, for sporadic ALS, the biomarker is selected from C9orf72 dipeptide repeat, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN, and GLUT1; wherein, for AD, the biomarker is selected from STOM and ANK1, and wherein, for PDD or PDnD, the biomarker is selected from CHL1, NELL2, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1.

[0077] Embodiment 22. The method of embodiment 21, wherein the biological fluid sample obtained is urine, whole blood, plasma or serum, cerebrospinal fluid (CSF), saliva or mucosa, such as urine, saliva, CSF or plasma, especially CSF or plasma. The biological fluid sample includes those in embodiment 3.

[0078] Embodiment 23. The method of Embodiment 21-Embodiment 22, wherein the human disease is sporadic ALS.

[0079] Embodiment 24. The method of Embodiment 21-Embodiment 22, wherein the human disease is AD.

[0080] Embodiment 25. The method of Embodiment 21-Embodiment 22, wherein the human disease is DLB, MSA, PDD or PDnD.

[0081] Example 26. The method of Example 21 to Example 25, further comprising thereafter treating the human subject for the human disease, or thereafter altering the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on detecting the presence of the isoxazole composition in the biological fluid, and optionally further comprising diagnosing the human disease in the human subject.

[0082] Embodiment 27. The method of embodiments 21 to 26 further comprises adding the polypeptide to the obtained biological fluid sample to facilitate detection by immunoassay, such as blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light scattering immunoassay, radiolabeled immunoassay, in particular ELISA or Western blot.

[0083] Embodiment 28. The method of Embodiment 27, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds a biomarker.

[0084] Example 29. The method of Example 28, wherein the human disease is sporadic ALS, and wherein the polypeptide used to detect sporadic ALS is an antibody to C9orf72 dipeptide repeat, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN, or GLUT1, such as C9orf72 dipeptide repeat.

[0085] Embodiment 30. The method of embodiment 28, wherein the human disease is AD, and wherein the polypeptide used to detect AD is an antibody against STOM or ANKL

[0086] Example 31. The method of Example 28, wherein the human disease is DLB, MSA, PDD or PDnD, and wherein the polypeptide used to detect PD is an antibody against CHL1, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 or RUVBL1.

[0087] Embodiment 32. The method of Embodiments 29 to 31, wherein the biological fluid is plasma or CSF.

[0088] Embodiment 33. The method of embodiment 27 to embodiment 32, wherein the method is ELISA, such as direct, indirect, sandwich or competitive ELISA.

[0089] Example 34. Using a biomarker in any of the methods of Examples 21 to 33 to detect a human disease.

[0090] Example 35. A method for detecting sporadic amyotrophic lateral sclerosis (ALS) in a human subject, comprising: detecting the presence of a dipeptide repeat protein from an obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject to obtain an obtained biological fluid sample.

[0091] Embodiment 36. The method of 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, especially CSF or plasma.

[0092] Embodiment 37. The method of Embodiment 35 to Embodiment 36, wherein the dipeptide repeat protein is selected from poly(GR), poly(GP) and poly(GA).

[0093] Example 38. The method of Example 35 to Example 37, further comprising thereafter treating the human subject for the human disease, or thereafter altering an existing treatment of the human subject for the human disease, such as treatment including drug therapy for the human disease, based on detecting the presence of the isoxazole composition in the biological fluid, and optionally further comprising diagnosing the human disease in the human subject.

[0094] Embodiment 39. The method of embodiments 35 to 38 further comprises adding the polypeptide to the obtained biological fluid sample to facilitate detection by immunoassay, such as blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light scattering immunoassay, radiolabeled immunoassay, in particular ELISA or Western blot.

[0095] Embodiment 40. The method of Embodiment 39, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds a biomarker.

[0096] Example 41. The method of Example 40, wherein the human disease is sporadic ALS, and wherein the polypeptide used to detect sporadic ALS is an antibody against a dipeptide repeat protein, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN, or GLUT1, such as a C9orf72 dipeptide repeat sequence.

[0097] Example 42. The method of Examples 39-41, wherein the biological fluid is plasma or CSF.

[0098] Embodiment 43. The method of embodiments 39-42, wherein the method is an ELISA, such as a direct, indirect, sandwich or competitive ELISA.

[0099] Example 44. Using a dipeptide repeat protein in any of the methods of Examples 35 to 43 to detect sporadic ALS.

[0100] Example 45. A method for detecting sporadic or SOD1 hereditary amyotrophic lateral sclerosis (ALS) in a human subject in the presymptomatic and prodromal stages, comprising: detecting the presence of a dipeptide repeat protein from an obtained biological fluid sample, and optionally obtaining a biological fluid sample from the subject to obtain an obtained biological fluid sample.

[0101] Embodiment 46. The method of 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, especially CSF or plasma.

[0102] Embodiment 47. The method of Embodiment 45 to Embodiment 46, wherein the dipeptide repeat protein is poly(GR).

[0103] Example 48. The method of Example 45 to Example 47, further comprising thereafter treating the human subject for the human disease, or thereafter altering the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on detecting the presence of the isoxazole composition in the biological fluid, and optionally further comprising diagnosing the human disease in the human subject.

[0104] Embodiment 49. The method of embodiments 45 to 48 further comprises adding the polypeptide to the obtained biological fluid sample to facilitate detection by immunoassay, such as blot assay, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), light scattering immunoassay, radiolabeled immunoassay, in particular ELISA or Western blot.

[0105] Embodiment 50. The method of Embodiment 49, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds a biomarker.

[0106] Example 51. The method of Example 50, wherein the polypeptide used to detect sporadic or SOD1 hereditary ALS is an antibody against a dipeptide repeat protein, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as the C9orf72 dipeptide repeat sequence.

[0107] Embodiment 52. The method of Embodiments 49 to 51, wherein the biological fluid is plasma or CSF.

[0108] Embodiment 53. The method of Embodiment 49 to Embodiment 52, wherein the method is ELISA, such as direct, indirect, sandwich or competitive ELISA.

[0109] Example 54. Using a dipeptide repeat protein in any of the methods of Examples 45 to 53 to detect sporadic or SOD1 hereditary ALS.

[0110] Embodiment 55. Use of any substance or composition in any of Embodiments 1 to 53 for diagnosing human diseases.

[0111] Method for diagnosing, monitoring and predicting disease progression of neurodegenerative diseases by detecting b-isox precipitate in patient plasma

[0112] The applicant used the small molecule compound b-isox to produce precipitation that can be observed with the naked eye in samples from ALS, AD, and PD patients, but not in samples from healthy subjects ( Figure 1 ). b-isox precipitation accurately distinguishes ALS patients from healthy subjects ( Figure 1 A).

[0113] Applicants have found that plasma (0.1 mL) is sufficient for this diagnosis ( Figure 1 C), and the reaction time of 60 minutes reached the optimal condition ( Figure 1 B). Our diagnostic accuracy for ALS, AD, and PD was 98.4%, 81.8%, and 92.8%, respectively ( Figure 1 D).

[0114] The size of b-isox deposits is negatively correlated with the functional score (ALSFRS-R), suggesting that b-isox deposits can be used not only for diagnosis but also for prediction of future progression of ALS disability ( Figure 1 E).

[0115] Methods for differential diagnosis and presymptomatic diagnosis, typing and drug response analysis of ALS by detecting b-isox-trapped proteins in CSF and plasma

[0116] The applicant has developed a new chemical ELISA method, called b-isox ELISA, to detect the level of specific proteins in b-isox precipitates ( Figure 2 a).

[0117] Applicants found that chemical ELISA detected the levels of specific b-isox-capture proteins tested in plasma and that there were significant differences between the neurodegenerative disease group and the healthy control group ( Figure 2 , 3 , 9 and 10).

[0118] Applicants have further found that the percentage of patients with high plasma misfolded proteins including SOD1, p-TDP-43 and C9orf72 corresponds closely to the proportion of patients whose inclusion bodies are known to be in the spinal cord from autopsy studies, such as Figure 2 These results demonstrate that the b-isox-based ELISA can detect real-time conformational changes of representative known pathogenic proteins, including TDP-43, SOD1, and C9orf72 dipeptide repeat (poly(GR)) proteins, in ALS patient plasma.

[0119] The applicant further identified new pathophysiological biomarkers of ALS through proteomic analysis of b-isox precipitates and further replicated and validated the identified candidate biomarkers in another group of patients ( Figure 3 and Figure 4 ). The levels of these newly identified biomarkers tested in plasma differed significantly between the disease groups and healthy individuals.

[0120] Edaravone, an antioxidant drug used clinically in ALS, reduced plasma PRDX2 levels but not TDP-43 or plasma GR repeat protein levels, revealing that the ineffectiveness of edaravone in clinical trials may be due to the inability to remove misfolded proteins and confirming the potential application of b-isoxELISA in drug response analysis in ALS ( Figure 6 ).

[0121] The data from all applicants suggest that plasma b-isox-trap proteins may serve as sensitive indicators of pathophysiological changes during the pathogenesis of neurodegenerative diseases and enable precision medicine approaches to neurodegenerative diseases.

[0122] Method for diagnosing PD by detecting b-isox capture protein in CSF and plasma

[0123] The applicant has developed a new chemical ELISA method, called b-isox ELISA, to detect the level of specific proteins in b-isox precipitates ( Figure 2 a).

[0124] Applicants found that chemical ELISA detected the levels of specific b-isox-capture proteins tested in plasma and that there were significant differences between the neurodegenerative disease group and the healthy control group ( Figure 8 and Fig. 9 ).

[0125] Applicants further identified novel biofluid biomarkers for PD by proteomic analysis of b-isox precipitates and further replicated and validated the identified candidate biomarkers in another cohort of patients ( Figure 8 and9 ). The levels of these new biomarkers tested in plasma differed significantly between the disease groups and healthy individuals.

[0126] Method for diagnosing AD by detecting b-isox capture protein in CSF and plasma

[0127] The applicant has developed a new chemical ELISA method, called b-isox ELISA, to detect the level of specific proteins in b-isox precipitates ( Figure 2 a).

[0128] Applicants found that chemical ELISA detected the levels of specific b-isox-capture proteins tested in plasma and that there were significant differences between the neurodegenerative disease group and the healthy control group ( Fig.10 ).

[0129] Applicants further identified novel AD biofluid biomarkers by proteomic analysis of b-isox precipitates and further replicated and validated the identified candidate biomarkers in another cohort of patients ( Fig.10 ). The levels of these new biomarkers tested in plasma differed significantly between the disease groups and healthy individuals.

[0130] Poly(GR) as a novel presymptomatic biomarker for inherited SOD1 and sporadic ALS

[0131] Aggregate deposition of dipeptide repeat proteins (poly GR, GP, or GA) is a hallmark of C9orf72-inherited ALS. Unexpectedly, GR repeat proteins appear early and remain at high levels in the plasma of 87% of ALS patients, which is the basis for ALS disease progression ( Figure 4 and Figure 7 a). These results suggest that poly(GR) proteins are increased in the plasma of patients with sporadic ALS, as hereditary patients account for only 10% of ALS. Our results reveal a novel pathophysiological role for plasma dipeptide repeat proteins (poly GR, GP, or GA).

[0132] No increase in poly(GR) levels was observed in SMA, AD, and PD ( Figure 7 b).

[0133] Applicants have discovered that increased poly(GR) protein appears in the prodromal phase of genetic individuals with C9orf72 mutations ( Figure 7 c), and presymptomatic in the SOD1 mouse model ( Figure 7d). Therefore, Applicants propose poly(GR) as a presymptomatic biomarker for C9orf72 ALS, sporadic ALS, and SOD1 inherited ALS.

[0134] In addition, the applicant also found that poly(GA) and poly(GP) were increased in the plasma of more than 70% of patients. The applicant suggested that poly(GA) and poly(GP) can be used not only as plasma biomarkers for C9orf72 hereditary ALS, but also as plasma biomarkers for the diagnosis of sporadic ALS ( Figure 2 ).

[0135] Description of materials and methods used in the examples

[0136] The following materials and methods were used in the examples described below.

[0137] Reagents and antibodies: b-isox was purchased from Sigma and Dalton and dissolved in dimethyl sulfoxide (DMSO). Primary antibody against SMN was purchased from BD Bioscience. Primary antibody against GFP was purchased from Roche. Primary antibodies against CA1 (#MBS1492724) and PRDX2 (#MBS7046127) were purchased from Mybiosource. Primary antibodies against MYL12B (#10324-1-AP), CD14 (#17000-1-AP), α-synuclein (#10842-1-AP), GR repeat protein (#23978-l-AP), phosphorylated TDP-43 (Ser 409 / 410) (#22309-1-AP), PFN1 (#11608-1-AP), NrCAM (#14255-A-AP), STOM (#120406-1-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 Cellsignaling.

[0138] b-isox precipitation: 10 mM biotinylated isoxazole was added to human plasma or CSF to a final concentration of 100 to 200 pM. The mixture was then incubated at 4°C for 60 min, centrifuged at 15,000 rpm at 4°C for 15 min, and the supernatant discarded. The diameter of the b-isox precipitate was measured.

[0139] b-isox precipitation and enzyme-linked immunosorbent assay (ELISA) Blood samples from patients and healthy controls were first collected through blood collection tubes. The tubes were centrifuged at 2200'g for 15 minutes. The resulting supernatant (upper layer) was used as the plasma sample. Before the immunoassay, 50-100 mL of plasma (or CSF) was gently mixed with 0.5-1 mL of b-isox by pipetting and rotated at 4°C for 1 hour. Wash with 200 mL of wash buffer (WB / (25 mM Tris, 150 mM NaCl; pH 7.2), 0.1% BSA, 0.05% -20) (do not let the wells dry). Add 100 μL of reaction mixture to each well and incubate at room temperature (RT) for 2 hours with shaking (~60 rpm). After the reaction is complete, wash 3 times with 200 μL of WB. Add 100 μL of primary antibody diluent (appropriately dilute the primary antibody in WB) and incubate at RT for 1 hour with shaking. In this step, a no primary antibody control should be included (add only antibody diluent to the sample well). Wash 3 times with 200 μL of WB. Add 100 μL of antibody diluent and appropriately diluted HRP-conjugated secondary antibody and incubate at RT for 1 hour with shaking. In this step, equilibrate the TMB substrate solution to room temperature. Wash 3 times with 200 μL of WB. Add 100 μL of TMB substrate solution to each microwell and incubate for 15-30 minutes until color develops. Stop the reaction by adding 100 μL of 2M sulfuric acid (or 2N hydrochloric acid). Measure the optical density at 450nm with a microplate reader.

[0140] Depletion of plasma misfolded proteins: Human plasma samples were incubated with A11 or B8H10 antibodies overnight at 4 degrees rotation (30 rpm). On the second day, the reaction samples were incubated with 5ul of magnetic beads conjugated to protein A / G beads (ThermoFisher) at room temperature for 1 hour with rotation (30 rpm). The mixture was then placed on a magnet for 3 minutes at room temperature to separate antibody-depleted plasma and antibody-captured proteins. The supernatant was collected and further tested by b-isoxELISA with specific biomarkers.

[0141] Example

[0142] Example 1: Analysis of the amount of b-isox precipitate in plasma from healthy controls and patients with ALS, AD and PD.

[0143] One milliliter of plasma from normal individuals and ALS patients was incubated with b-isox and centrifuged to reduce the cross-β prion-like LC protein ( Figure 1 a), b-isox precipitates can be observed with the naked eye in samples from patients but not in samples from healthy subjects ( Figure 1a). The optimal condition for b-isox precipitation is to incubate 100 μl of plasma with b-iox for 60 min ( Figure 1 b and Figure 1 c).

[0144] Figure 1 d shows the statistical analysis of the size of b-isox precipitates in 100 μl of plasma from normal subjects and ALS, AD, and PD patients. The b-isox precipitation accuracy rates for ALS, AD, and PD were 98.4%, 81.8%, and 92.8%, respectively.

[0145] Follow-up analysis of two ALS patients showed that the size of b-isox deposits was negatively correlated with functional score (ALSFRS-R) ( Figure 1 e). These results suggest that b-isox precipitation may be used not only for diagnosis but also for prediction of future progression of ALS disability.

[0146] Example 2: Differential diagnosis of ALS by b-isox ELISA.

[0147] To detect the levels of specific low complexity (LC) proteins in b-isox precipitates, we developed a chemical ELISA method called b-isox ELISA. The schematic diagram of b-isox ELISA is shown in Figure 2 As shown in a. Plasma was mixed with b-isox to generate b-isox-bound complexes, and then we used streptavidin to capture the b-isox-bound complexes, followed by traditional ELISA using specific antibodies against the target of interest.

[0148] First, we analyzed the levels of b-isox-captured SOD1 in the plasma of control, ALS mice with the SOD1 G93A mutation, and SMA mice by b-isox-based ELISA ( Figure 2 b) We found that the increase in SOD1 levels in the precipitates isolated from the plasma of ALS mice correlated with the time period during which misfolded SOD1 protein aggregated in ALS mice. The levels of SOD1 in the plasma of SMA mice did not change.

[0149] Consistently, we examined the SOD1 levels in the plasma of patients with inherited SOD1 G93A mutation and found that the plasma SOD1 levels were significantly elevated in patients with inherited ALSFRS-R 43 compared with controls ( Figure 2 c).

[0150] Furthermore, in an analysis of 120 ALS patients, the approximately 10% of patients with high plasma SOD1 closely approximates the proportion of patients known to have SOD1 inclusions in the spinal cord from autopsy studies. Figure 2 As shown in d.

[0151] Next, we measured the levels of p-TDP-43 in precipitates isolated from ALS patient plasma by b-isox ELISA. Consistently, we found that p-TDP-43 patients correlated with the reported incidence of TDP-43 in ALS ( Figure 2 e).

[0152] In patients with high plasma p-TDP-43 levels, we found that plasma GR repeat protein levels were also elevated, but TDP-43 and SOD1 levels were not elevated ( Figure 2 f).

[0153] A longitudinal study showed that both plasma p-TDP-43 levels and the size of b-isox precipitates decreased during ALS progression ( Figure 2 g).

[0154] We next examined plasma GR repeat protein levels in normal controls and ALS patients. The positive rate of patients with elevated GR repeat protein was as high as ∼87.5%, covering the sporadic ALS group, which has not been reported ( Figure 2 h).

[0155] Representative profile of ALS patients showing that in patients with high plasma GR repeat protein levels, plasma p-TDP-43 and SOD1 proteins were also not elevated ( Figure 2 i).

[0156] Figure 2 j shows the longitudinal tracking of the association between plasma GR repeat protein levels, the size of b-isox precipitates, and ALSFRS-R.

[0157] Example 3: Identification and validation of new pathophysiological biomarkers for ALS.

[0158] By using proteomic analysis of b-isox-generated precipitates recovered from CSF of patients with ALS, AD, or PD, we discovered several potential new pathophysiological biomarkers involved in the pathogenesis of ALS, AD, or PD. A comprehensive flowchart of the experiment is shown in Figure 3 a. Protein identification of b-isox-captured precipitates from plasma of healthy individuals and ALS, AD, or PD patients was performed by LC-Ms / Ms, and the differentially expressed proteins were classified into membrane trafficking, exosome, metabolism, spliceosome, peptides and inhibitors, and chromosome and related proteins based on the enriched gene ontology (GO) terms.

[0159] We replicated and validated the identified candidate biomarkers in another cohort of patients and confirmed CA1, CD14, MYL12B, PRDX2, STOM, GLUT-1, SMN, β-actin (ACTB), PFN1, poly(GA), and poly(GP) as pathophysiological biomarkers of ALS ( Figure 3 b) Levels of the nine b-isox-trapped proteins tested in plasma differed significantly between the disease groups and healthy individuals.

[0160] Unexpectedly, the applicants found that the dipeptide repeat proteins poly(GR), poly(GA), and poly(GP) were increased in the plasma of more than 80% of patients, indicating that these proteins can be used as biomarkers not only for C9orf72-inherited ALS, but also for sporadic ALS diagnosis ( Figure 2 and Figure 3 ).

[0161] Example 4: Association between plasma b-isox-trap protein and ALS disability.

[0162] To explore the relationship between proteinopathy of regulatory proteins / risk proteins and disability progression in ALS patients, we analyzed the correlation between pathogenic variant levels and the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score. Plasma pathogenic variant levels were detected, including p-TDP-43, GR repeat protein, MYL12B, STOM, SOD1, CD14, CA1, PRDX2, and ANXA5. Patients were divided into four stages according to the functional score ( Figure 4 a). ALSFRS-R is divided into four groups: stage I (40-48 points), stage II (30-39 points), stage III (20-29 points), and stage IV (10-19 points). The percentages above represent the proportion of patients with increased biomarkers.

[0163] In addition to the presence of high SOD1 in the early stages of ALS, a subgroup with low SOD1 emerged at later stages. Given that SOD1 inclusions were detected in motor neurons of sALS patients carrying C9ORF72 repeat expansions without SOD1 mutations, we infer that the induction of SOD1 misfolding is a common downstream event in ALS progression ( Figure 4 a).

[0164] In addition, PRDX2, MYL12B, and CD14 showed similar trends, which decreased after stage II (score 30-39) ( Figure 4 b).

[0165] High GR repeat proteins are maintained during the development of disability ( Figure 4 a and Figure 4 c).

[0166] Notably, the presence of high ANXA5 protein in the plasma of ALS patients only in the early stages of ALS reflects the occurrence of neuronal renewal, which may trigger the onset of ALS disease ( Figure 4 d).

[0167] Example 5: Longitudinal tracking of b-isox-trapped proteins in ALS plasma.

[0168] We performed b-isox ELISA analysis of ALS progression in 23 participants who were followed for up to 3.5 years with repeated clinical examinations. We observed that b-isox-trapped proteins in patient plasma changed dynamically during disease progression of ALS in individual patients. Figure 5 Two examples are shown in FIG.

[0169] Example 6: Analysis of drug response to edaravone in ALS

[0170] By monitoring the real-time drug response of two ALS patients treated with edaravone using b-isox ELISA, we found that edaravone treatment decreased plasma PRDX2 and CD14 but had no significant effect on plasma GR repeat protein ( Figure 6 ). Edaravone is an antioxidant that could explain the reduction in PRDX2. We speculate that the ineffectiveness of edaravone in ALS clinical trials may be due to a failure to reduce the pathological burden of misfolded GR repeat proteins.

[0171] Example 7: poly(GR) is a very early biomarker for SOD1 hereditary and sporadic ALS

[0172] Of all the ALS biomarkers we obtained from b-isox precipitation analysis, only GR repeat protein appears early and remains at high levels in the plasma of patients as ALS disease progresses ( Figure 4 and Figure 7 a). However, increased poly(GR) protein is absent in SMA, AD, and PD, suggesting that GR repeat protein is a specific biomarker for ALS ( Figure 7 b).

[0173] Single females with inherited C9orf72 repeats showed increases in poly(GA), CD14, and poly(GR) proteins during the prodromal period, but no changes in p-TDP-43 and SOD1 ( Figure 7 c).

[0174] Indeed, we observed that plasma GR repeat protein increased presymptomatically in SOD1 G93A mice and appeared earlier than plasma misfolded SOD1 ( Figure 7d). These results suggest that the GR repeat protein could serve as a very early biomarker, not only as a prodromal biomarker for inherited C9orf72 ALS, but also for sporadic ALS and other types of inherited ALS such as SOD1 and TDP-43 mutations.

[0175] Example 8: Identification and validation of plasma biomarkers for PD by b-isox ELISA

[0176] We analyzed PD patients by b-isox ELISA and showed significant quantitative differences between chemical precipitates from PD patients and healthy individuals. Using proteomic analysis, we comprehensively analyzed precipitates isolated from CSF of PD patients and obtained a small number of candidate biomarkers for PD.

[0177] As expected, we confirmed elevated levels of synuclein, p-TDP-43, PRDX2, CHL1, NrCAM, STOM, CA1, CD14, ANXA5, NELL2, and RUVBL1 in the plasma of PD patients by b-isox-ELISA ( Figure 8 ).

[0178] Example 9: Molecular analysis 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 ( Fig. 9 ).

[0179] Example 10: Identification and validation of plasma biomarkers for AD by b-isox ELISA

[0180] We analyzed AD patients by b-isox ELISA and showed significant quantitative differences between the chemical precipitates of AD patients and healthy individuals. Using proteomic analysis, we comprehensively analyzed precipitates isolated from CSF of AD patients.

[0181] As expected, we confirmed elevated levels of amyloid β, tau, p-TDP-43, TDP-43ANK1, and STOM in the plasma of AD patients by b-isox-ELISA ( Fig.10 a).

[0182] Notably, ANK1 and STOM showed to be more sensitive than amyloid β by b-isox-ELISA ( Fig.10 b).

[0183] References

[0184] 1. Professor et al., Conformational diseases, The Lancet, 1997, vol. 350, pp. 134-138.

[0185] 2. Kiernan et al., Amyotrophic lateral sclerosis, 2017, The Lancet. Volume 390, Pages 2084-2098.

[0186] 3. Rosen et al., Cu / Zn superoxide dismutase gene mutations associated with familial amyotrophic lateral sclerosis, Nature, 1993, Vol. 362(6415), pp. 59-62.

[0187] 4. Neumann et al., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, Science, 2006, vol. 314, pp. 130–133.

[0188] 5. Wang et al., TDP-43: an emerging player in neurodegenerative diseases, 2008, Trends in Molecular Medicine, vol. 14, pp. 479-485.

[0189] 6. DeJesus-Hernandez et al., Expanded GGGGCC hexanucleotide repeats in the noncoding region of C9ORF72 cause chromosome 9p-linked FTD and ALS. Neuron, 72, 2011, 245–56.

[0190] 7. Achmitz et al., Emerging perspectives on C9ORF72ALS / FTD dipeptide repeat proteins, 2021,

[0191] The Lancet, vol. 397, pp. 1577–1590.

[0192] 8. Scheltens et al., Alzheimer's disease, 2021, The Lancet, 397, 1577-1590.

[0193] 9. Braak et al., Staging of brain pathology associated with sporadic Parkinson's disease. Neurobiology of Aging, 24(2), 197-211, 2003.

[0194] 10. Wang et al., Native TDP-43 C-terminal self-interaction inhibits its degradation and leads to early proteinopathy, Nature Communications, 2012, vol. 3, p. 766.

[0195] 11. Kato et al., Cell-free formation of RNA granules: low-complexity sequence domains form dynamic fibers within hydrogels, Cell, 149, 753–767, 2012.

[0196] 12. Han et al., Cell-free formation of RNA granules: combining RNA to identify features and components of cellular assemblies, Cell, 149, 768–779, 2012.

[0197] 13. Brangwynne et al., “Germinal P particles are droplets localized by controlled dissolution / condensation”, Science, 324, 1729–1732 (2009).

[0198] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.The specification and examples are to be considered as exemplary only.

Claims

1. A method for detecting a human disease in a human subject, comprising, optionally obtaining a biological fluid sample from said subject, adding an isoxazole to the obtained biological fluid sample to form a biological fluid isoxazole composition in the biological fluid sample, and The biological fluid is detected for the presence of the isoxazole composition.

2. The method according to claim 1, wherein: The isoxazole 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 an analog thereof, in particular biotin-isoxazole, very in particular biotin-isoxazole.

3. The method according to claims 1 to 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, in particular CSF or plasma.

4. The method according to claims 1 to 3, wherein: The human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious diseases, Huntington's disease, schizophrenia, aging-related diseases or protein diseases, especially wherein the human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), Lewy body dementia (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy with neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

5. The method of claims 1 to 4, further comprising thereafter treating the human subject for the human disease, or thereafter changing the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on said detecting the presence of said biological fluid isoxazole composition, and optionally further comprising diagnosing said human disease in said human subject.

6. The method according to claims 1 to 5, wherein: The concentration of isoxazole in the biological fluid sample ranges from 0.075 mM to 0.225 mM, preferably from 0.100 mM to 0.200 mM.

7. The method according to claims 1 to 6, wherein: The biological fluid isoxazole composition is in the precipitate.

8. The method according to claim 7, wherein: The human disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA) or frontotemporal dementia (FTLD).

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

10. The method according to claim 9, wherein: The human disease is ALS, such as sporadic ALS.

11. The method of claims 7 to 10, further comprising monitoring the size of the precipitate to monitor the progression of ALS in the subject.

12. The method according to claims 1 to 6, further comprising adding a polypeptide to the biological fluid isoxazole composition in the biological fluid 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, in particular an ELISA or a Western blot.

13. The method according to claim 12, wherein: The human conformational disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD), Parkinson's disease non-dementia (PDnD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA) and marginal dominant age-related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological changes (LATE-NC), stroke, cerebral amyloid angiopathy (CAA) or frontotemporal dementia (FTLD).

14. The method according to claims 12 to 13, wherein: The polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds the biological fluid isoxazole composition.

15. The method according to claim 14, wherein: The human disease is ALS, and wherein the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeat, PFN1, PRDX2, phosphorylated TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB or GLUT1, such as SOD1, MYL12B, CD14 and p-TDP-43, and in particular SOD1 and p-TDP-43.

16. The method according to claim 14, wherein: The human disease is AD, and wherein the polypeptide used to detect AD is an antibody against APP, phosphorylated TDP-43, TDP-43, Tau, STOM or ANK1.

17. The method according to claim 14, wherein: The human disease is PD, and wherein the polypeptides used to detect PD are antibodies against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 and RUVBL1.

18. The method according to claims 15 to 17, wherein: The biological fluid is plasma or CSF.

19. The method according to claims 12 to 18, wherein: The method is an ELISA, such as a direct, indirect, sandwich or competitive ELISA.

20. Use of an isoxazole in a method according to any of claims 1 to 19 for detecting a human disease.

21. A method for detecting a human disease in a human subject, comprising, detecting the presence of a biomarker from the obtained biological fluid sample, and optionally obtaining a biological fluid sample from said subject to obtain said obtained biological fluid sample, and in, The human disease is selected from the group consisting of sporadic amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease dementia (PDD) and Parkinson's disease without dementia (PDnD), Wherein, for sporadic ALS, the biomarker is selected from C9orf72 dipeptide repeat, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN and GLUT1 Wherein, for AD, the biomarker is selected from STOM and ANK1, and Wherein, for DLB, MSA, PDD or PDnD, the biomarker is selected from CHL1, NELL2, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 and RUVBL1.

22. The method according to claim 21, wherein: The biological fluid sample obtained is urine, whole blood, plasma or serum, cerebrospinal fluid (CSF), saliva or mucosa, such as urine, saliva, CSF or plasma, especially CSF or plasma.

23. The method according to claims 21 to 22, wherein: The human disease is ALS.

24. The method according to claims 21 to 22, wherein: The human disease is AD.

25. The method according to claims 21 to 22, wherein: The human disease is DLB, MSA, PDD or PDnD.

26. The method of claims 21 to 25, further comprising thereafter treating the human subject for the human disease, or thereafter changing the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on said detecting the presence of said biological fluid isoxazole composition, and optionally further comprising diagnosing said human disease in said human subject.

27. The method according to claims 21 to 26, further comprising adding a polypeptide to the obtained biological fluid sample to facilitate detection by an immunoassay, such as a western blot assay, a chemiluminescent immunoassay, an enzyme-linked immunosorbent assay (ELISA), a light scattering immunoassay, a radiolabeled immunoassay, in particular an ELISA or a Western blot.

28. The method according to claim 27, wherein: The polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds to the biomarker.

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

30. The method of claim 28, wherein: The human disease is AD, and wherein the polypeptide used to detect AD is an antibody against STOM or ANKL.

31. The method of claim 28, wherein: The human disease is DLB, MSA, PDD or PDnD, and wherein the polypeptide used to detect PD is an antibody against CHL1, NrCAM, ANK1, STOM, PRDX2, CA1, CD14 or RUVBL1.

32. The method according to claims 29 to 31, wherein: The biological fluid is plasma or CSF.

33. The method according to claims 27 to 32, wherein: The method is an ELISA, such as a direct, indirect, sandwich or competitive ELISA.

34. Use of any method according to claims 21 to 33 for detecting a biomarker of said human disease.

35. A method for detecting amyotrophic lateral sclerosis (ALS) in a human subject, comprising, The presence of the dipeptide repeat protein is detected from an obtained biological fluid sample, and optionally the biological fluid sample is obtained from said subject to obtain said obtained biological fluid sample.

36. The method of claim 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, in particular CSF or plasma.

37. The method according to claims 35 to 36, wherein: The dipeptide repeat protein is selected from the group consisting of poly(GR), poly(GP) and poly(GA).

38. The method of claims 35 to 37, further comprising thereafter treating the human subject for the human disease, or thereafter changing the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on said detecting the presence of said biological fluid isoxazole composition, and optionally further comprising diagnosing said human disease in said human subject.

39. The method according to claims 35 to 38, further comprising adding a polypeptide to the obtained biological fluid 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, in particular an ELISA or a Western blot.

40. The method of claim 39, wherein: The polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds to the biomarker.

41. The method of claim 40, wherein: The human disease is sporadic ALS, and wherein the polypeptide for detecting sporadic ALS is an antibody against a dipeptide repeat protein, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as a C9orf72 dipeptide repeat sequence.

42. The method according to claims 39 to 41, wherein: The biological fluid is plasma or CSF.

43. The method according to claims 39 to 42, wherein: The method is an ELISA, such as a direct, indirect, sandwich or competitive ELISA.

44. Use of a dipeptide repeat protein in any method according to claims 35 to 43 for detecting sporadic ALS.

45. A method for detecting sporadic or SOD1 inherited amyotrophic lateral sclerosis (ALS) in a human subject in the presymptomatic and prodromal stages, comprising, The presence of the dipeptide repeat protein is detected from an obtained biological fluid sample, and optionally the biological fluid sample is obtained from said subject to obtain said obtained biological fluid sample.

46. ​​The method of claim 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, in particular CSF or plasma.

47. The method according to claims 45 to 46, wherein: The dipeptide repeat protein is poly(GR).

48. The method of claims 45 to 47, further comprising thereafter treating the human subject for the human disease, or thereafter changing the human subject's existing treatment for the human disease, such as treatment including drug therapy for the human disease, based on said detecting the presence of said biological fluid isoxazole composition, and optionally further comprising diagnosing said human disease in said human subject.

49. The method according to claims 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, a chemiluminescent immunoassay, an enzyme-linked immunosorbent assay (ELISA), a light scattering immunoassay, a radiolabeled immunoassay, in particular an ELISA or a Western blot.

50. The method of claim 49, wherein: The polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof that binds to the biomarker.

51. The method of claim 50, wherein: The polypeptide for detecting sporadic or SOD1 hereditary ALS is an antibody against dipeptide repeat proteins, PRDX2, CA1, MYL12B, CD14, ANXA5, STOM, SMN or GLUT1, such as C9orf72 dipeptide repeat sequences.

52. The method of claims 49 to 51, wherein: The biological fluid is plasma or CSF.

53. The method of claims 49 to 52, wherein: The method is an ELISA, such as a direct, indirect, sandwich or competitive ELISA.

54. Use of a dipeptide repeat protein in any method according to claims 45 to 53 for detecting sporadic or SOD1 hereditary ALS.

55. Use of any of the substances or compositions for diagnosing a human disease according to any one of claims 1 to 53.

Images