Detection of disease state macromolecules binding to normal macromolecules as a biomarker for disease identification

Abstract

In one aspect, the present disclosure provides a method of detecting a presence or absence of a biomarker for a disease in the sample, wherein the biomarker comprises: a) a complex of physiologically active target macromolecules or a fragment or portion thereof and target macromolecules that are not physiologically active; b) a conformation of the physiologically active macromolecules or fragment thereof when the physiologically active target macromolecules or the fragment or portion thereof is a complex with a non- physiologically active target macromolecule; c) the conformation of physiologically active target macromolecules or a portion or fragment thereof in a PAT-Tau complex; d) the conformation of non-physiologically active target macromolecules or a portion or fragment thereof in a PAT-Tau complex; or e) a combination of a), b), c), d) and / or e).

Description

DETECTION OF DISEASE STATE MACROMOLECULES BINDING TO NORMAL MACROMOLECULES AS A BIOMARKER FOR DISEASE IDENTIFICATIONCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 631,943, filed April 9, 2024, U.S. Provisional Application No. 63 / 575,585, filed April 5, 2024, and U.S. Provisional Application No. 63 / 589,273, filed October 10, 2023, each of which is entirely incorporated herein by reference.BACKGROUND

[0002] Some diseases can be caused by a misfolding, mutation, or other alteration to a normal protein to create a disease protein. In some embodiments, a disease can be detected through the detection of a disease protein. In some embodiments, patients who carry one or two copies of a disease gene can develop a disease as a result of the misfolded, mutated, or altered protein. The physiological changes produced by misfolded, mutated, or altered protein can initiate years or even decades before noticeable physiological symptoms manifest.

[0003] Early detection of a disease offers several benefits, including timely interventions potentially delaying or mitigating the symptoms associated with the disease and allowing patients to access clinical trials and supportive care services. Developing reliable diagnostic tests for early disease detection is a significant research focus. While several successful diagnostic tests have been developed, they tend to be more effective in diagnosing symptomatic patients. There is a notable absence of minimally invasive screening tools for asymptomatic to early-stage detection of diseases using easily accessible specimens.

[0004] Developing diagnostics for the pre-symptomatic stage of diseases or related pathology can be complex due to the subtle nature of early changes in a subject. Additionally, the variability in individual age-based, gender-based, and ethnicity-based responses to the disease can further complicate the identification of reliable biomarkers. Pursuing effective and minimally invasive diagnostic tools for early-stage disease or related pathology detection is crucial to ongoing research. Early stages of disease or related pathology involves the presence of biomarkers at extremely low concentrations. Detecting and / or characterizing these trace amounts of biomarkers requires highly sensitive detection methods which can also eliminate or minimize influence by other substances or interferences present in the sample.SUMMARY

[0005] Disclosed herein in some embodiments are methods and systems for biomarker detection for disease identification. The compositions and methods described herein can provide forenhanced ability to characterize a biomarker as physiologically active and / or detect a biomarker (e.g., a physiologically active biomarker) in a sample. In an aspect, the present disclosure provides a method of detecting a physiologically active target macromolecule, the method comprising: a) providing a sample from a subject; and b) detecting a presence or absence of a biomarker for a disease in the sample, wherein the biomarker comprises: i) a physiologically active target macromolecule (PATM) or a portion or fragment thereof specific to a disease macromolecule pathology and the disease pathology, wherein the physiologically active target macromolecule comprises a macromolecule that is at least partially hyperphosphorylated, misfolded, post translationally modified, insoluble, truncated, mutated, present in an aggregated form, present in a tangle, or any combination thereof when compared to a non-physiologically active macromolecule (NPATM); ii) a conformation of the PATM or a portion or fragment thereof specific to a pathology of the macromolecule and the disease pathology; iii) a complex of PATM or a portion or fragment thereof and non-physiologically active target macromolecule (NPATM) or a portion or fragment thereof (PATM-NPATM complex); iv) a conformation of the PATM or a portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM-NPATM complex; v) a conformation of the NPATM or a portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM-NPATM complex; vi) a binding of NPATM or a portion or fragment thereof to PATM or a portion or fragment thereof; or vii) any combination of i) - vi). In some embodiments, the PATM or the portion or fragment thereof comprises NPATM or a portion or fragment thereof that is at least partially hyperphosphorylated (HPT), mis-folded, post translationally modified, insoluble, truncated, mutated, present in an aggregated form, present in a tangle, or any combination thereof. In some embodiments, the PATM or the portion or fragment thereof comprises a target protein or a portion or fragment thereof that comprises an ability to bind the NPATM or a portion or fragment thereof. In some embodiments, the NPATM or the portion or fragment thereof comprises a macromolecule or a portion or fragment thereof that is not associated with a disease state. In some embodiments, the subject is pre-symptomatic for, or suspected of having, the disease and the method further comprises: detecting a presence of a disease pathology or a prognosis of the subject developing the disease when the biomarker for the disease is present in the sample; or detecting an absence of the disease pathology or a prognosis of the subject not developing the disease when the biomarker for the disease is absent in the sample. In some embodiments, the detecting comprises detecting the PATM-NPATM complex by detecting a binding of a capture moiety that is specific to: a) the PATM-NPATM complex, b) the conformation of the PATM or the portion or fragment thereof in the PATM-NPATM complex,c) the conformation of target macromolecule or a portion or fragment thereof in a PATM- NPATM complex, or d) a combination of a), b), and / or c). In some embodiments, the capture moiety that is specific to the PATM-NPATM complex comprises an anti-hyperphosphorylated antibody against a specific phosphorylation site, an antibody against a conformation of the PATM or the portion or fragment thereof specific to target macromolecule pathology and disease pathology, an antibody against the PATM-NPATM complex, an antibody against a conformation of the PATM or the portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM-NPATM complex, NPATM or a portion or fragment thereof, an anti-PATM polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, a F(ab) antibody, a F(ab')2 antibody, chimeric antibody, a chimeric ’ab'2 antibody fragment, a molecular imprinted polymer (MIP), an aptamer, an alpaca nanobody, a llama derived nanobody, a portion or fragment of any of these, or any combination thereof. In some embodiments, the capture moiety comprises two or more capture moieties. In some embodiments, the recombinant antibody comprises a recombinantly tagged protein or a portion or fragment thereof. In some embodiments, the recombinantly tagged protein or the portion or fragment thereof comprises an anti-tag antibody. In some embodiments, the anti-tag antibody comprises an anti-recombinant tag antibody. In some embodiments, the anti-recombinant tag comprises an anti -V 5 tag, an anti-6His tag, an anti-8His tag, an anti -HA, an anti -FLAG, an anti- c-Myc, an anti-GST, an anti-fusion domain, or any combination thereof. In some embodiments, the anti-V5 tag antibody comprises a monoclonal anti-V5 tag antibody.

[0006] In some embodiments, the detecting comprises: capturing the PATM-NPATM complex; disrupting the PATM-NPATM complex to release the NPATM or the portion or fragment thereof; and detecting the NPATM or the portion or fragment thereof. In some embodiments, the detecting comprises: capturing the target macromolecule or the portion or fragment thereof on a substrate; and determining whether a captured target macromolecule or the portion or fragment thereof is PATM or a portion or fragment thereof by exposing it to a NPATM or a portion or fragment thereof, wherein the NPATM complexes with the captured target macromolecule when the captured target macromolecule is PATM. In some embodiments, the NPATM or the portion or fragment thereof is labeled with a label. In some embodiments, the label comprises a fluorophore, a stable isotope, a mass tag, a horse radish peroxidase (HRP), an alkaline phosphatase (ALP), a luciferase, a chemiluminescent substrate, an electrochemiluminescence substrate, a peptide, an oligonucleotide, a recombinant tag, an HA tag (YPYDVPDYA), a FLAG tag (DYKDDDDK), a c-Myc tag (EQKLISEEDL), a GST tag, a tandem mass tagged antibody, a tandem mass tagged peptide, a stable isotope labelled antibody or peptide, a fragment or portionof any of these, or any combination thereof. In some embodiments, the chemiluminescent substrate comprises an isoluminol, a luminol, an acridinium ester, an ABEI, or any combination thereof. In some embodiments, the electrochemiluminescence substrate comprises a ruthenium. In some embodiments, the peptide comprises an SmTrip9, an SmTriplO, or a combination thereof. In some embodiments, the detecting comprises detecting a conjugate or detection reagent in a sandwich immunoassay or an inhibition immunoassay. In some embodiments, the conjugate comprises an antibody or capture moiety that directly or indirectly generates assay signal, fluorescence, HRP fluorescence, ALP fluorescence, light, bioluminescence, color, mass signature, immuno-PCR signal, or any combination thereof. In some embodiments, the conjugate comprises an HRP, an ALP, a Luciferase, a fluorophore, a chemiluminescent substrate, an electrochemiluminescence substrate, a bioluminescent substrate, an oligonucleotide for immune-PCR, or any combination thereof. In some embodiments, the method further comprises the chemiluminescent substrate, wherein the chemiluminescent substrate comprises an isoluminol, a luminol, an acridinium ester, an ABEI, a HRP substrate, an ALP substrate, an immuno-PCR oligonucleotide labeled antibody or peptide, stable isotope labelled antibodies or peptides, peptides labeled with light isotopes, tandem mass tagged antibodies or peptides, a fragment or portion of any of these, or any combination thereof. In some embodiments, the conjugate comprises the chemiluminescence substrate, wherein the electrochemiluminescence substrate comprises ruthenium. In some embodiments, the conjugate comprises the bioluminescent substrate, wherein the bioluminescent substrate comprises SmTrip9 or SmTriplO. In some embodiments, an anti-PATM capture moiety is labeled with SmTrip9 and an anti-PATM-NPATM capture moiety is labeled with SmTriplO. In some embodiments, an anti- PATM capture moiety is labeled with SmTriplO and an anti-PATM-NPATM capture moiety is labeled with SmTrip9. In some embodiments, the detecting of the biomarker comprises detecting a specific phosphorylated epitope of the Target macromolecule. In some embodiments, the method further comprises measuring an assay signal, dose, or PAT or PATM-NPATM concentration. In some embodiments, the detecting of the biomarker comprises detecting through electrophoresis, immunoblotting, immunoprecipitation, autoradiography, mass spectrometry, proteomics, protein separation, western blotting, protein identification, immunoassay, light scattering, spectrometry, calorimetry, biolayer interferometry (BLI), surface plasmon resonance (SPR), or other label-free optical techniques for measuring biomolecular interactions, or any combination thereof. In some embodiments, the detecting of the biomarker comprises detecting through MS, time-of-flight mass spectrometry (TOF-MS), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), LC-MS, or LC-MS / MS. In someembodiments, the detecting of the biomarker comprises binding the biomarker to biomarker capture particles comprising a biomarker capture moiety. In some embodiments, the method further comprises quantitating the amount of biomarker that binds to the biomarker capture particles. In some embodiments, the biomarker capture particles are coated with at least a first type of antibody specific to the first type of epitope and a second type of antibody specific to the second type of epitope. In some embodiments, the biomarker capture particles comprise agglutination particles, wherein the agglutination particles aggregate upon binding to the biomarker. In some embodiments, the biomarker capture particles comprise microparticles. In some embodiments, the biomarker capture particles comprise beads. In some embodiments, the biomarker capture particles comprise a metal. In some embodiments, the biomarker capture particles are magnetic. In some embodiments, the biomarker capture particles comprise: a plurality of magnetic beads, and a plurality of non-magnetic beads, wherein the plurality of magnetic beads is larger in size than the plurality of non-magnetic beads, and wherein one or more of the pluralities of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker. In some embodiments, a concentration of the plurality of nonmagnetic beads decreases upon removal of the complex. In some embodiments, the method further comprises measuring the binding of the biomarker with the detection antibody. In some embodiments, the method further comprises comparing the binding of the biomarker with the detection antibody to a standard curve. In some embodiments, the standard curve is generated from biomarker capture particles bound to known amounts of the biomarker. In some embodiments, the biomarker capture particles are coated with an antibody, and wherein the standard curve is generated from known amounts of liquid or lyophilized peptide that is added to the biomarker capture particles. In some embodiments, the method further comprises quantitating the biomarker. In some embodiments, the sample comprises a first sample and the method further comprises monitoring the biomarker over time to determine an increase or decrease in an amount of the biomarker in a second sample of the subject, relative to the amount of biomarker in the first sample. In some embodiments, the method comprises diagnosing the subject with the disease based at least in part on detecting: i) the PATM or a fragment or portion thereof specific to target macromolecule pathology and disease pathology; ii) the conformation of the PATM or a fragment or portion thereof specific to target macromolecule pathology and disease pathology; iii) the complex of PATM or a fragment or portion thereof and non-physiologically active target macromolecule or a fragment or portion thereof (PATM- NPATM complex); iv) the conformation of the NPATM or a fragment or portion thereof when the NPATM or the fragment or portion thereof is in the PATM-NPATM complex; v) theconformation of the PATM or a fragment or portion thereof when the PATM or the fragment or portion thereof is in the PATM-NPATM complex; vi) the binding of NPATM or a fragment or portion thereof to PATM or a fragment or portion thereof; or vii) any combination of i) - vi). In some embodiments, the method further comprises administering a treatment to the subject for the disease. In some embodiments, the target macromolecule pathology comprises an insoluble target macromolecule aggregate, a neurofibrillary tangle, a PATM or a fragment or portion thereof, a PATM-NPATM complex, or any combination thereof. In some embodiments, the method further comprises determining a course of a disease progression based on the quantity of the biomarker detected, the quantity of the amount of target macromolecule or the fragment or portion thereof that is physiologically active, or a combination thereof. In some embodiments, a signal to noise ratio of the detection is more than about 3.0. In some embodiments, the macromolecule comprises a human prion protein (PrP), a tau protein, a transactive response DNA binding protein 43 (TDP-43), an M protein, an alpha-synuclein (a- syn), a glial fibrillary acidic protein (GFAP), a beta-amyloid 1-42 (AB (1-42)), a neurofilament light chain, a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, an HGF protein, a CTSD protein, a amylin, an Islet Amyloid Polypeptide (IAPP), a huntingtin (HTT) protein, a p53 protein, a tumor suppressor protein, an oncoprotein, an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2) protein, an ezrin protein, a portion or fragment of any of these, or any combination thereof. In some embodiments, the disease comprises a Creutzfeldt-Jakob disease, a motor neuron disease, an Amyotrophic Lateral Sclerosis, a Multiple Myeloma, a Parkinson's Disease, a Type II diabetes, a Huntington's disease, a cancer, a Basal cell carcinoma, an Alzheimer’s disease, or any combination thereof. In some embodiments, a) the target macromolecule comprises a human prion protein (PrP), a tau protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active human prion protein (PrP), a physiologically active tau protein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active human prion protein (PrP), a non-physiologically active tau protein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Creutzfeldt-Jakob disease.

[0007] In some embodiments, a) the target macromolecule comprises a transactive response DNA binding protein 43 (TDP-43), a portion or fragment thereof, or any combination thereof; b) the PATM comprises a physiologically active TDP-43, a portion or fragment thereof, or any combination thereof; c) the NPATM comprises a non-physiologically active TDP-43, a portion or fragment thereof, or any combination thereof; and d) the disease comprises an AmyotrophicLateral Sclerosis (ALS). In some embodiments, a) the target macromolecule comprises an M protein, a portion or fragment thereof, or any combination thereof; b) the PATM comprises a physiologically active M protein, a portion or fragment thereof, or any combination thereof; c) the NPATM comprises a non-physiologically active M protein, a portion or fragment thereof, or any combination thereof; and d) the disease comprises a Multiple Myeloma. In some embodiments, a) the target macromolecule comprises an alpha-synuclein (a-syn), a glial fibrillary acidic protein (GFAP), a beta-amyloid 1-42 (AB (1-42)), a tau, a neurofilament light chain, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active alpha-synuclein (a-syn), a physiologically active glial fibrillary acidic protein (GFAP), a physiologically active beta-amyloid 1-42 (AB (1-42)), a physiologically active tau, a physiologically active neurofilament light chain, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non- physiologically active alpha-synuclein (a-syn), a non-physiologically active glial fibrillary acidic protein (GFAP), a non-physiologically active beta-amyloid 1-42 (AB (1-42)), a non- physiologically active tau, a non-physiologically active neurofilament light chain, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Parkinson’s disease. In some embodiments, a) the target macromolecule comprises a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, an HGF protein, a CTSD protein, an amylin, an Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active follistatin protein, a physiologically active insulin-like growth factor binding protein 2, a physiologically active FABP4 protein, a physiologically active HGF protein, a physiologically active CTSD protein, a physiologically active amylin, a physiologically active Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active follistatin protein, a non-physiologically active insulin-like growth factor binding protein 2, a non-physiologically active FABP4 protein, a non-physiologically active HGF protein, a non-physiologically active CTSD protein, a non-physiologically active amylin, a non-physiologically active Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a diabetes. In some embodiments, the diabetes comprises a Type II diabetes. In some embodiments, a) the target macromolecule comprises a glial fibrillary acidic protein, a beta-amyloid 1-42 (AB (1-42)), a tau protein, huntingtin (HTT) protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active p53 protein, a physiologically active tumor suppressor protein, a physiologically active oncoprotein, a portion or fragment of any ofthese, or any combination thereof; c) the NPATM comprises a non-physiologically active tumor suppressor protein, a non-physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Huntington's disease. In some embodiments, a) the target macromolecule comprises a p53 protein, a tumor suppressor protein, an oncoprotein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active p53 protein, a physiologically active tumor suppressor protein, a physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active p53 protein, a non- physiologically active tumor suppressor protein, a non-physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a cancer. In some embodiments, a) the target macromolecule comprises an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2) protein, an ezrin protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active asprosin protein, a physiologically active suprabasin protein, a physiologically active cyclooxygenase-2 (COX-2) protein, a physiologically active ezrin protein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active asprosin protein, a non-physiologically active suprabasin protein, a non-physiologically active cyclooxygenase-2 (COX-2) protein, a non-physiologically active ezrin protein, a portion or fragment of any of these, or any combination thereof, and d) the disease comprises a basal cell carcinoma. In some embodiments, a) the target macromolecule comprises a Tau protein or a portion or fragment thereof; b) the PATM comprises a physiologically active Tau (PAT) protein or a portion or fragment thereof; c) the NPATM comprises a non-physiologically active Tau (NPAT) protein or a portion or fragment thereof, and d) the disease comprises an Alzheimer’s disease. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau!81, a pTau!99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, a pTau422, or any combination thereof. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau231. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau217. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau231 and a pTau231. In some embodiments, the PAT protein or the portion or fragment thereof comprises a microtubule-binding region (MTBR) of tau comprising: (a) MTBR R1 residues 244-274, MTBR R2 residues 275-305, MTBR R3 residues 306-336, MTBR R4 residues, or any combination thereof; (b) MTBR sites implicated in tau aggregation comprising residues 275-280 (vqiink), 306-311 (vqivyk), or a combination thereof; (c) residues 225-242 near residue 243 and Rl; (d)residues 226-264 near residue 260 within Rl; (e) R1 residues 256-273 (vkskigstenlkhqpggg), R2 residues 287-304 (vqskcgskdnikhvppgg), R3 residues 318-335 (vtskcgslgnihhkpggg), R4 residues 350-364 (vqskigsldnithvpggg), and combinations thereof; or (f) Rl residues 268-271 (hqpg), R2 residues 299-302 (hvpg), R3 residues 330-333 (hkpg), R4 residues 362-365 (hvpg), or any combination thereof. In some embodiments, the PAT protein or the portion or fragment thereof comprises a specific phosphorylation site at Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, Ser422, or any combination thereof. In some embodiments, the non-PAT protein or the portion or fragment thereof comprises recombinant human Tau-441, or a fragment or portion of tau441 peptide selected from a full length peptide, a truncated peptide, a mutated peptide, a modified recombinant amino acid sequence in any position(s) in the peptide, amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising a tau aggregation sequence at the begging of R2 (VQIINK tau aggregation site) and / or R3 (VQIVYK tau aggregation site), a portion or fragment of any of these, or any combination thereof. In some embodiments, the detecting of the biomarker comprises detecting a specific phosphorylated epitope of Tau. In some embodiments, the specific phosphorylated epitope of Tau comprises anti-Thr!81, anti-Ser!99, anti-Ser202, anti-Thr205, anti-Thr212, anti- Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti-Ser404, or anti- Ser422, and the conjugate is against a different phosphorylated or non-phosphorylated Tau epitope. In some embodiments, the antibody or antibody binding fragment comprises a tau-441 peptide, or a fragment or portion of tau441 peptide comprising at least one of MTBR R1-R4. In some embodiments, treatment comprises a medicament or disease modifying therapy (DMT) that targets amyloid pathology (amyloid plaque), tau pathology, neuroregeneration, cognitive function, or any combination thereof. In some embodiments, a treatment can comprise administering an anti-sense oligonucleotide (ASO) to a subject in need thereof. In some embodiments, a treatment can comprise administering a pharmaceutically effective amount of TRIM21 to a subject in need thereof. In some embodiments, the tau pathology comprises an insoluble tau aggregate, a neurofibrillary tangle, a PAT protein or a fragment or portion thereof, a PAT-Tau complex, or any combination thereof. In some embodiments, a medicament can comprise a galantamine, a rivastigmine, a donepezil, an immunotherapy, or any combination thereof. In some embodiments, the capture moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a heavy chaincomplementarity determining region 3 (CDR-H3) comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3). In some embodiments, the VH comprises a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5). In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4). In some embodiments, the VL comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9). In some embodiments, the VL comprises a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8). In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7). In some embodiments, the capture moiety comprises: a CDR-H3 comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3); a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5); a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4); a CDR-L3 comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9); a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8); and a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7). In some embodiments, the VH comprises a sequence with at least 80% sequence identity to the sequence QVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10). In some embodiments, the VL comprises a sequence with at least 80% sequence identity to the sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11). In some embodiments, the VH comprises the amino acid sequence of QVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10). In some embodiments, the VL comprises the amino acid sequence of DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11). In some embodiments, the antibody or antibody binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3). In some embodiments, the VH comprises a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5). In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of GYTFTSH(SEQ ID NO: 4). In some embodiments, the VL comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9). In some embodiments, the VL comprises a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8). In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7). In some embodiments, the antibody or antibody binding fragment comprises: a CDR-H3 comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3); a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5); a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4); a CDR-L3 comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9); a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8); and a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7). In some embodiments, the VH comprises a sequence with at least 80% sequence identity to the sequence QVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10). In some embodiments, the VL comprises a sequence with at least 80% sequence identity to the sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11). In some embodiments, the VH comprises the amino acid sequence of QVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10). In some embodiments, the VL comprises the amino acid sequence of DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11). In some embodiments, the detecting comprises: (a) contacting the sample comprising the biomarker with a composition comprising a first reagent and a second reagent, wherein the first reagent deactivates one or more interference enzymes, wherein the second reagent solubilizes the biomarker; and (b) contacting the sample with a plurality of particles, wherein the plurality of particles comprises a capture moiety to bind the biomarker.

[0008] In some embodiments, the present disclosure provides a method, comprising: (a) contacting a sample comprising a biomarker with a composition comprising a first reagent and a second reagent, wherein the first reagent deactivates one or more interference enzymes, wherein the second reagent solubilizes the biomarker; and (b) contacting the sample with a plurality ofparticles, wherein the plurality of particles comprises a capture moiety to bind the biomarker. In some embodiments, the method further comprises, prior to (b), contacting the sample with an additional plurality of particles, wherein the additional plurality of particles binds one or more interference molecules in the sample. In some embodiments, the one or more interference enzymes comprise one or more endogenous phosphatase. In some embodiments, the second reagent further reduces non-specific binding of the biomarker to the additional plurality of particles. In some embodiments, the composition further comprises a third reagent, wherein the third reagent blocks one or more biotin binding moieties. In some embodiments, the detecting comprises: contacting a sample comprising one or more phosphorylated tau proteins with a composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein.

[0009] In some embodiments, the present disclosure provides a method, comprising: contacting a sample comprising one or more phosphorylated tau proteins with a composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein. In some embodiments, the detecting comprises detecting a PATM in a sample, comprising: (a) capturing the PATM with a capture moiety to form a capture moiety -PATM complex; (b) contacting the capture moiet -PATM complex with a non-PAT (NPATM) or a portion or fragment thereof, to form a capture moiety-PATM-NPATM complex; (c) contacting the capture moiety-HPT-PATM-NPATM complex with a detection moiety, wherein the detection moiety binds to the capture moiety-PATM-NPATM complex to form a capture moiety-HPT-PATM-NPATM complex-detection moiety complex; and (d) detecting the capture moiety-HPT-PATM-NPATM complex-detection moiety complex.

[0010] In some embodiments, the present disclosure provides a method of detecting a hyperphosphorylated tau (HPT) protein in a sample, comprising: (a) capturing the HPT protein with a capture moiety to form a capture moiety-HPT complex: (b) contacting the capture moiety-HPT complex with a non-hyperphosphorylated tau (nTau) protein or a portion or fragment thereof, to form a capture moiety -HPT-nTau complex; (c) contacting the capture moiety -HPT -nTau complex with a detection moiety, wherein the detection moiety binds to thecapture moiety -HPT-nTau complex to form a capture moiety-HPT-nTau complex-detection moiety complex; and (d) detecting the capture moiety-HPT-nTau complex-detection moiety complex.

[0011] In some embodiments, the present disclosure provides a composition, comprising: (a) a first reagent configured to deactivate one or more interference enzymes; (b) a second reagent configured to solubilize a biomarker; (c) a third reagent configured to block biotin binding moieties; or (d) any combination of (a)-(c).

[0012] In some embodiments, the present disclosure provides a kit, comprising: (a) a first reagent configured to deactivate one or more interference enzymes; (b) a second reagent configured to solubilize a biomarker; (c) a third reagent configured to block biotin binding moieties; or (d) any combination of (a)-(c). In some embodiments, the kit further comprises Tris-HCl, NaCl, or sodium azide, or any combination thereof.

[0013] In some embodiments, the present disclosure provides a composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein.

[0014] In an aspect, the present disclosure provides a composition comprising a monoclonal antibody that is specific to a PATM-NPATM complex. In an aspect, the present disclosure provides a composition comprising a monoclonal antibody that is specific to the conformation of PATM or a fragment or portion thereof in a PATM-NPATM complex. In some embodiments, the monoclonal antibody is a human monoclonal antibody, an animal antibody, or a humanized animal antibody.

[0015] In an aspect, the present disclosure provides a method comprising: a) providing a sample from a subject; and b) detecting a presence or absence of a biomarker for Alzheimer’s disease in the sample, wherein the biomarker comprises: (i) a physiologically active Tau (PAT) protein or a portion or fragment thereof specific to tau pathology and Alzheimer's disease pathology; ii) a conformation of the PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer's disease pathology; iii) a complex of PAT protein or a portion or fragment thereof and non-hyperphosphorylated tau (non-PAT) protein or a portion or fragment thereof (PAT-Tau complex); iv) a conformation of the PAT protein or a portion or fragment thereof when the PAT protein or the portion or fragment thereof is in the PAT-Tau complex; v) a binding of non-PAT protein or a portion or fragment thereof to PAT protein or a portion or fragment thereof; or vi) any combination of i) - v).

[0016] In some embodiments, the PAT protein or a portion or fragment thereof comprises Tau or a portion or fragment thereof that is hyperphosphorylated (PAT), mis-folded, post translationally modified, insoluble, present in an aggregated form, present in a tangle, or any combination thereof. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTaul81, a pTaul99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, a pTau422, or any combination thereof. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau231. In some embodiments, the PAT protein or the portion or fragment thereof comprises a pTau217. In some embodiments, the PAT protein or the portion or fragment thereof comprises both a pTau217 and a pTau231. In some embodiments, the PAT protein or the portion or fragment thereof comprises a microtubule-binding region (MTBR) of tau comprising (a) MTBR Rl residues 244-274, MTBR R2 residues 275-305, MTBR R3 residues 306-336, MTBR R4 residues, or combinations thereof; (b) MTBR sites implicated in tau aggregation comprising residues 275-280 (VQIINK), 306-311 (VQIVYK), or combination thereof; (c) residues 225-242 near residue 243 and Rl; (d) residues 226-264 near residue 260 within Rl; (e) R1 residues 256- 273 (VKSKIGSTENLKHQPGGG), R2 residues 287-304 (VQSKCGSKDNIKHVPPGG), R3 residues 318-335 (VTSKCGSLGNIHHKPGGG), R4 residues 350-364 (VQSKIGSLDNITHVPGGG), or combinations thereof; or (f) Rl residues 268-271 (HQPG), R2 residues 299-302 (HVPG), R3 residues 330-333 (HKPG), R4 residues 362-365 (HVPG), or combinations thereof. In some embodiments, the PAT protein or a portion or fragment thereof comprises Tau that comprises an ability to bind non-PAT protein or a portion or fragment thereof. In some embodiments, non-PAT protein or a portion or fragment thereof comprises protein that is not associated with a disease state. In some embodiments, the subject is pre- symptomatic for Alzheimer’s disease and the method further comprises: detecting a presence of Alzheimer’s disease pathology or a prognosis of developing Alzheimer’s disease in the subject when the biomarker for Alzheimer’s disease is present in the sample; or detecting an absence of Alzheimer’s disease pathology or a prognosis of not developing Alzheimer’s disease in the subject when the biomarker for Alzheimer’s disease is absent in the sample. In some embodiments, the detecting comprises detecting the PAT-Tau complex by detecting a binding of a capture moiety that is specific to: a) the PAT-Tau complex, b) the conformation of PAT protein or a portion or fragment thereof in a PAT-Tau complex, c) the conformation of Tau or a portion or fragment thereof in a PAT-Tau complex, or d) a combination of a), b), and / or c). In some embodiments, the capture moiety that is specific to the PAT-Tau complex comprises an anti-hyperphosphorylated antibody against a specific phosphorylation site, an antibody against aconformation of the PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer's disease pathology, an antibody against the PAT-Tau complex, an antibody against a conformation of the PAT protein or a portion or fragment thereof when the PAT is in the PAT- Tau complex, non-PAT, an anti-PAT polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, a Fab, a Fab'2, chimeric antibody, a chimeric Fab'2 antibody fragment, a molecular imprinted polymer (MIP), an aptamer, an alpaca nanobody or a llama derived nanobody. In some embodiments, the capture moiety comprises two or more capture moieties. In some embodiments, the specific phosphorylation site comprises Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, Ser422, or any combination thereof. In some embodiments, the non-PAT or the portion or fragment thereof comprises recombinant human Tau-441, or a fragment or portion of tau441 peptide such as full length peptide, truncated peptide, modified recombinant amino acid sequence in any position(s) in the peptide, amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising the tau aggregation sequence at the begging of R2 (VQIINK tau aggregation site) and or R3 (VQIVYK tau aggregation site), or any combination thereof. In some embodiments, the recombinant antibody comprises a recombinantly tagged protein or a portion or fragment thereof. In some embodiments, the recombinantly tagged protein or the portion or fragment thereof comprises an anti-tag antibody. In some embodiments, the anti-tag antibody comprises an anti -recombinant tag antibody. In some embodiments, the recombinant tag comprises a V5 tag, polyHis (e.g., 6His, 8His, or longer polyHis) tag, HA, FLAG, c-Myc, GST, or fusion domains, or any combination thereof. In some embodiments, the anti-V5 tag antibody comprises a monoclonal anti-V5 tag antibody. In some embodiments, the detecting comprises: capturing a PAT-Tau complex; disrupting the PAT-Tau complex to release non-PAT protein or the portion or fragment thereof; and detecting the non-PAT protein protein or the portion or fragment thereof. In some embodiments, the detecting comprises: capturing the Tau protein or the portion or fragment thereof on a substrate; and determining whether a captured Tau protein or the portion or fragment thereof is PAT protein or a portion or fragment thereof by exposing it to a non-PAT protein or a portion or fragment thereof, wherein the non-PAT protein complexes with the captured Tau when the captured Tau protein is PAT. In some embodiments, the non-PAT or the portion or fragment thereof is labeled. In some embodiments, the label comprises a fluorophore, a stable isotope, a mass tag, horse radish peroxidase (HRP), alkaline phosphatase(ALP), a Luciferase, a chemiluminescent substrate such as isoluminol, luminol, acridinium ester, or, ABEI, an HRP substrate such as TMB Substrate Solution, an enhanced HRP detection substrate such as FemtoMax™ Super Sensitive HRP Substrate by Rockland Immunochemicals, Chemiluminescent Substrate Solution (HRP Marker) by Aero Biosystems, QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit, 1-Step™ ABTS Substrate Solution, or SuperSignal™ West Femto Maximum Sensitivity Substrate by ThermoFisher, Novex™ HRP Chromogenic Substrate (TMB) or Novex™ ECL Chemiluminescent Substrate Reagent Kit by Invitrogen, or ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) substrate, CHMI Chemiluminescent Ultra Sensitive HRP Microwell Substrate, or LUMI Chemiluminescent Super Sensitive HRP Microwell and / or Membrane Substrate by Suromids, or Lumi-Phos HRP (PS- atto) by Lumigen, or other similar enhanced HRP substrates, an ALP chemiluminescence substrate such as Lumi-Phos 530, Lumigen APS-5 , or Lumi-Phos Plus by Lumigen, an ALP fluorescence substrate such as AttoPhos® Substrate by Promega, an electrochemiluminescence substrate such as ruthenium, a peptide such as Lumit® SmTrip9 or SmTriplO, oligonucleotide, a recombinant tag such as a polyhistidine tag (e.g., His-tag, an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag), a V5 tag (a small epitope tag comprising a sequence including IPNPLLGLD (SEQ ID NO: 28), which can be placed by molecular cloning at the N- or C-terminus of a protein of choice), a HA tag (YPYDVPDYA; SEQ ID NO: 24), a FLAG tag (DYKDDDDK; SEQ ID NO: 25), a c-Myc tag (EQKLISEEDL; SEQ ID NO: 26), a GST tag (26 kDa sequence of 211 amino acids), or a combination thereof. In some embodiments, the detecting comprises detecting a conjugate or detection reagent in a sandwich immunoassay or an inhibition immunoassay. In some embodiments, the detecting can comprise using biolayer interferometry (BLI) and / or a biosensor. These methods may detect binding of normal protein (e.g., non-diseased protein) to disease protein. As another example, BLI and surface plasmon resonance (SPR) may be used as label-free optical technologies to measure one or more biomolecular interactions. In some embodiments, conjugate comprises an antibody or capture moiety that directly or indirectly generates assay signal, fluorescence, HRP fluorescence, ALP fluorescence, light, bioluminescence, color, mass signature, immuno-PCR signal, or a combination thereof. In some embodiments, the conjugate comprises an HRP, an ALP, a Luciferase, a fluorophore, a chemiluminescent substrate, an electrochemiluminescence substrate, a bioluminescent substrate, or any combination thereof. In some embodiments, the chemiluminescent substrate comprises an isoluminol, a luminol, an acridinium ester, an ABEI, a HRP substrate, an ALP substrate, animmuno-PCR oligonucleotide labeled antibody or peptide, stable isotope labelled antibodies or peptides (e.g., peptides labeled with light isotopes (1H,12C,14N, and / or16O, peptides labeled with heavy isotopes (2H,13C,15N, and / or18O), e.g., peptide labeled with light isotopes combined with peptides labeled with heavy isotopes (2H,13C,15N, and / or18O) prior to mass spectrometry (MS) and the relative peak intensity of the two forms provides an accurate indication of the relative levels of each peptide in the sample), tandem mass tagged antibodies or peptides (e.g., six varieties of TMT: TMTzero, a non-isotopically substituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; 10-plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 plex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero), are, or any combination thereof. In some embodiments, the electrochemiluminescence substrate comprises ruthenium. In some embodiments, the bioluminescent substrate comprises SmTrip9 or SmTriplO. In some embodiments, an anti-PAT capture moiety is labeled with SmTrip9 and an anti-PAT-Tau capture moiety is labeled with SmTriplO. In some embodiments, an anti-PAT capture moiety is labeled with SmTriplO and an anti-PAT-Tau capture moiety is labeled with SmTrip9. In some embodiments, the detecting of the biomarker comprises detecting a specific phosphorylated epitope of Tau. In some embodiments, the specific phosphorylated epitope of Tau comprises anti-Thr!81, anti-Ser!99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti- Ser262, anti-Ser396, anti-Ser404, or anti-Ser422, and the conjugate is against a different phosphorylated or non-phosphorylated Tau epitope. In some embodiments, the method further comprises measuring an assay signal, dose, or PAT or PAT-Tau concentration. In some embodiments, the detecting of the biomarker comprises detecting through electrophoresis, immunoblotting, immunoprecipitation, autoradiography, mass spectrometry, proteomics, protein separation, western blotting, protein identification, immunoassay, light scattering, spectrometry, calorimetry, or any combination thereof. In some embodiments, the detecting of the biomarker comprises detecting through mass spectrometry (MS), time-of-flight mass spectrometry (TOF- MS), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), liquid chromatography- mass spectrometry (LC-MS), or LC-MS / MS. In some embodiments, the detecting of the biomarker comprises binding the biomarker to biomarker capture particles comprising a biomarker capture moiety.

[0017] In some embodiments, the method further comprises quantitating the amount of biomarker that binds to the biomarker capture particles. In some embodiments, the biomarkercapture particles are coated with at least a first type of antibody specific to the first type of epitope and a second type of antibody specific to the second type of epitope. In some embodiments, the biomarker capture particles comprise agglutination particles, wherein the agglutination particles aggregate upon binding to the biomarker. In some embodiments, the biomarker capture particles comprise microparticles. In some embodiments, biomarker capture particles comprise beads. In some embodiments, the biomarker capture particles comprise a metal. In some embodiments, the biomarker capture particles are magnetic. In some embodiments, the biomarker capture particles comprise: a plurality of magnetic beads, and a plurality of non-magnetic beads, wherein the plurality of magnetic beads are larger in size than the plurality of non-magnetic beads, and wherein one or more of the pluralities of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker. In some embodiments, a concentration of the plurality of non-magnetic beads decreases upon removal of the complex. In some embodiments, the biomarker capture moiety comprises an antibody or antibody binding fragment. In some embodiments, the antibody binding fragment is selected from the group consisting of autoantibodies, therapeutic antibodies, immunoglobulin classes, immunoglobulin subclasses, circulating antibodies, secretory antibodies, alpaca derived nanobodies, and animal derived antibodies. In some embodiments, the antibody or antibody binding fragment comprises a tau441 peptide, or a fragment or portion of tau441 peptide comprising at least one of MTBR R1-R4. In some embodiments, the sample comprises a biological fluid sample. In some embodiments, the biological fluid sample comprises blood, plasma, serum, a reconstituted dry blood spot, capillary collection, saliva, saline oral rinse, stool, urine, tears, cerebrospinal fluid, or any combination thereof. In some embodiments, the method further comprises removing interferences from the sample prior to the detecting of the presence or absence of the biomarker. In some embodiments, the removing interferences from the sample comprises cleaning the sample by removing the interferences from the sample with the interference capture particles. In some embodiments, the interference capture particles comprise an interference capture moiety that interacts with a sample interference. In some embodiments, the sample interference is HAMA, RF, an autoantibody to Tau or pTau, a Macro-Tau complex of Tau, pTau, or both Tau and pTau with autoantibodies, anti-streptavidin interference, antibiotin interference, anti-PEG interference (or PEOn), anti-detection moiety interference (such as human anti-ALP, anti-ruthenium, anti-fluorescein, anti-ABEI interference), free biotin interference, or combination thereof. In some embodiments, the subject is a human. In some embodiments, the method further comprises binding the captured biomarker with a detection antibody. In some embodiments, the method further comprises measuring the binding of adetection antibody. In some embodiments, the method further comprises comparing the binding of the detection antibody to a standard curve. In some embodiments, the standard curve is generated from biomarker capture particles bound to known amounts of the biomarker. In some embodiments, the biomarker capture particles are coated with 77G7 antibody, and wherein the standard curve is generated from known amounts of Tau441-V5 or liquid or lyophilized Tau441- V5 peptide, or fragment or portion of tau441 peptide comprising at least one of MTBR R1-R4, that is added to the biomarker capture particles. In some embodiments, the method further comprises quantitating the biomarker. In some embodiments, the biomarker capture particles are coated with RD-073 antibody, ADx204 antibody, HT7 antibody, BT2 antibody, 77G7 antibody, or any combination thereof. In some embodiments, the sample comprises a first sample and the method further comprises monitoring the biomarker over time to determine an increase or decrease in an amount of the biomarker in a second sample of the subject, relative to the amount of biomarker in the first sample. In some embodiments, the method comprises diagnosing the subject with Alzheimer’s disease based at least in part on detecting: i) the physiologically active Tau (PAT) protein specific to tau pathology and Alzheimer’s disease pathology; ii) the conformation of the PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer’s disease pathology; iii) the complex of PAT protein or a portion or fragment thereof and non-physiologically active tau (PAT-Tau complex); iv) the conformation of the PAT protein or a portion or fragment thereof when the PAT protein or a portion or fragment thereof is in the PAT-Tau complex; v) the binding of non-PAT protein or a portion or fragment thereof to PAT protein or a portion or fragment thereof; or vi) any combination of i) - v). In some embodiments, the method further comprises administering a treatment to the subject for Alzheimer’s disease. In some embodiments, the treatment comprises a medicament or disease modifying therapy (DMT) that targets amyloid pathology (amyloid plaque), tau pathology, neuroregeneration, cognitive function, or any combination thereof. In some embodiments, the tau pathology comprises an insoluble tau aggregate, a neurofibrillary tangle, a PAT protein or a fragment or portion thereof, a PAT-Tau complex, or any combination thereof. In some embodiments, the medicament comprises a galantamine, a rivastigmine, a donepezil, or any combination thereof. In some embodiments, the medicament is a tau-directed therapy such as targeting tau PTMs (Tideglusib [NP031112, Nypta®, Zentylor™, GSK-3P inhibitor, NP12], Lithium, Sodium selenate [VEL015], LY3372689, and Salsalate), aggregation inhibitors (LMTM [TRxO237, LMT-X, Methylene Blue, TAI], ACI3024 [Tau MorphomerTM], and Curcumin [Diferuloylmethane, Longvida™]), tau expression inhibitors (BIIB080 [IONIS-MAPTRX, ISIS 814907]), microtubule stabilizers (Davunetide [NAP, AL-108] and Epothilone [BMS-241027]),active immunization (AADvacl [Axon peptide 108 conjugated to KLH] and ACI-35), passive immunization (RG7345 [RO6926496], Gosuranemab [BIIB092, BMS-986168, IPN007], Semorinemab [R07105705, MTAU9937A, RG6100], Zagotenemab [LY3303560], JNJ63733657, Bepranemab [UCB0107, UCB 0107, Antibody D], PNT001, BIIB076 [NI-105, 6C5huIgGl / l], Tilavonema [C2N8E12, ABBV8E12, HJ9.3], E2814, and Lu AF87908). In some embodiments, the method further comprises determining a course of a disease progression based on the quantity of the biomarker detected, the quantity of the amount of tau that is physiologically active, or a combination thereof. In some embodiments, a signal to noise ratio of the detection is more than about 3.0.

[0018] In some embodiments, the present disclosure provides a composition comprising a monoclonal antibody that is specific to a PAT-Tau complex. In some embodiments, the present disclosure provides a composition comprising a monoclonal antibody that is specific to the conformation of PAT protein or a portion or fragment thereof in a PAT-Tau complex. In some embodiments, the monoclonal antibody is an animal monoclonal antibody such as mouse, rabbit, or sheep. In some embodiments, the monoclonal antibody is a human monoclonal antibody or humanized mouse monoclonal antibody.INCORPORATION BY REFERENCE

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0021] FIG. 1 shows the adverse effects of interferences and sample matrix on patient samples, a challenge faced by assay manufacturers and CLIA labs when developing and commercializing diagnostics tests. Heterophilic interference and sample matrix effects can cause false negatives and / or positives, skewing the testing results.

[0022] FIG. 2 shows an example process for processing a patient sample.

[0023] FIG. 3 shows that the application of interference capture particles (or clean beads, used interchangeably herein) to a patient sample can lead to a significant reduction in sampleinterferences.

[0024] FIG. 4 shows the high-performance liquid chromatography (HPLC) purification of biotinylated monomeric IgG for clean and capture bead preparation. IgG as such or upon biotinylation often form aggregates that can interfere with antigen capture efficiency or introduce non-specific signals. The monomeric IgGs were selectively purified to prepare interference capture particles.

[0025] FIG. 5 shows a homogeneous, no-wash immunoassay that detects a given analyte in test samples.

[0026] FIG. 6 shows an exemplary 4-20% gradient Tris-Glycine gel with protein files of the SOR and treated sample.

[0027] FIG. 7A shows amyloid beta 1-40 (AB40) response curve.

[0028] FIG. 7B shows amyloid beta 1-42 (AB42) response curve.

[0029] FIG. 7C shows phosphorylated Tau-181 (pTaul81) response curve.

[0030] FIG. 8 shows that the immunoassay detection range of AB40 of an immunoassay was from 0.120pg / mL (120 fg / mL) to 2.5 ng / mL.

[0031] FIG. 9 shows exemplary detection results for samples before and after cleaning and enrichment.

[0032] FIG. 10A, FIG. 10B, and FIG. 10C show AB40, pTaul81, and summation of AB40 and pTaul81 levels in AD patients’ SOR samples compared to those of normal, healthy controls.

[0033] FIG. 11 shows different amino acid epitopes of Tau that can be targeted by different antibodies.

[0034] FIG. 12 shows the sequences of the binding epitopes for antibody 77G7.

[0035] FIG. 13 shows amino acid (a.a.) sequence of 255-269 (MTBR1) and 286-300(MTBR2) of the binding epitopes across the Tau441 peptide for antibody 77G7 with OD450 values.

[0036] FIG. 14 shows a.a. sequence of 317-331 (MTBR3) and 349-363 (MTBR4) ofthe binding epitopes across the Tau441 peptide for antibody 77G7 with OD450 value.

[0037] FIG. 15 shows an exemplary process for detecting the AD biomarker of PAT or a portion or fragment thereof binding to non-PAT protein or a portion or fragment thereof as a biomarker of AD pathology and related tauopathies.

[0038] FIG. 16 shows the fluorescence signal as a function of the Tau441-V5 concentration measured on the biomarker capture particles or Tau441-V5 concentration measured after its elution and neutralization from the biomarker capture particles.

[0039] FIG. 17 shows the dose response fluorescence signal of different concentrations of Tau441-V5 captured by 77G7 antibody coated biomarker capture particles read at 15 minute intervals.

[0040] FIG. 18 shows the fluorescence signal read at 60 minutes using different concentrations (dilutions) of the anti-V5-AP conjugate (PANEL A: the % coefficient of variation (% CV) and PANEL B: signal to noise ration at 60 minute of incubation with substrate).

[0041] FIG. 19 shows exemplary results of the assay protocol to only detect PAT binding to non-PAT protein in AD Plasma (positive relative absorptions) with and not detect PAT binding to non-PAT protein in Normal Plasma (HRP substrate background signal relative absorptions) with 100% accuracy.

[0042] FIGS. 20A-20D show amino acid sequences of an exemplary antibody. FIG. 20A shows a sequence of a light version of 77G7 (with 219 a.a.).

[0043] FIG. 20B shows a sequence of a heavy version of 77G7 (with 443 a. a.).

[0044] FIG. 20C shows the CDR and framework region annotations with Chothia scheme for the light version of 77G7 shown in FIG. 20A.

[0045] FIG. 20D shows the CDR and framework region annotations with Chothia scheme for the heavy version of 77 G7 shown in FIG. 20B.

[0046] FIG. 21 illustrates an assay method for detection of Alzheimer's disease (AD) biomarkers.

[0047] FIG. 22A shows detection accuracy using a tau assay described herein. FIG. 22B shows detection accuracy of FIG. 22A using a tau assay described herein in a different y scale. FIG. 22C shows detection accuracy of FIG. 22A using a tau assay described herein in different y scales.

[0048] FIG. 23 shows a multi-day assay method for detection of AD biomarkers.

[0049] FIG. 24 shows the quantitative measure was amount of immunoassay signal, as measured by relative light unit (RLU).

[0050] FIG. 25 shows detection accuracy using a tau assay described herein. The raw data was transformed from relative light units (RLU) to ratio data using a calibrator or assay cutoff or cut point.DETAILED DESCRIPTIONDefinitions

[0051] Unless defined otherwise, all terms of art, notations and other technical and scientificterms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some embodiments, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0052] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0053] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0054] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing”, as used interchangeably herein, generally refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0055] The terms “subject,” “individual,” or “patient”, as used interchangeably herein, generally refer to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject can be diagnosed or suspected of being at high risk for a disease. In some embodiments, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0056] The term “i« vivo”, as used herein, generally refers to an event that takes place in a subject’s body.

[0057] The term “ex vivo”, as used herein, generally refers to an event that takes placeoutside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “m vitro" assay.

[0058] The term “m vitro", as used herein, generally refers to an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0059] The terms, “analyte”, “target”, and “biomarker”, as used interchangeably herein, generally refer to a molecule or complex to be detected and / or quantitated. Non-limiting examples of biomarkers comprise, but are not limited to, polypeptides (e.g., proteins, phosphorylated or other post-translationally modified forms of a protein, antibodies, etc.), antigens, small molecules, and nucleic acids (e.g., DNAs, RNAs, mRNAs, ribosomal RNAs, microRNAs, transcription factor binding sites, genomic DNAs or RNAs, etc.). In some embodiments, a biomarker can comprise a binding activity, conformation, or interaction between two or more molecules or complexes.

[0060] The terms “Alzheimer’s disease biomarker” or “AD biomarker” generally refer to any biomarker that can be detected in a sample from a subject that would indicate the subject has Alzheimer’s disease. In some embodiments, an AD biomarker can be detected in a subject who is pre-symptomatic of Alzheimer’s disease. In some embodiments, an AD biomarker can comprise a physiologically active tau protein (PAT) or a fragment or portion thereof, a binding of PAT to normal Tau, a complex of PAT to normal Tau, a conformation of PAT when complexed to normal Tau, a conformation of normal Tau when complexed to PAT, or any combination thereof.

[0061] The term “Tau protein”, “tau protein”, “Tau”, or “tau” as used interchangeably herein generally refers to full length proteins, portions thereof, fragments thereof, peptides, or amino acid sequences of the Tau protein, which can comprise a microtubule-associated protein that forms insoluble filaments that accumulate as neurofibrillary tangles in Alzheimer’s disease (AD) and related tauopathies. In some embodiments, a Tau protein can comprise a recombinant Tau peptide. In some embodiments, a Tau protein can comprise a Tau 441 peptide. In some embodiments, a Tau protein can comprise full length, truncated, a recombinant amino acid sequence changed in any position, an amino acid modification in any position(s), or any combination thereof, as long as the Tau protein can still bind to PAT. In some embodiments, only 1 or more of the Microtubule Binding (MTBR) regions are required such as Rl, or R2, orR3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or regions between R1 and R2, between R2 and R3, or regions between R1 / R2 and R2 / R3 combined such as KHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLK which can represent a truncated normal tau peptide with the tau binding domains, residues or epitopes required for HPT binding to normal Tau to form insoluble tau aggregates and neurofibrillary tangles between MTBR R1 / R2 and MTBR R2 / R3.

[0062] The term “macromolecule” generally refers to a protein, nucleic acid, synthetic polymer, or combination thereof.

[0063] The term “protein” generally refers to a polymer of amino acids and can include full length proteins, peptides, or shorter fragments thereof.

[0064] The term “physiologically active target macromolecule” or “PATM” generally refers to a macromolecule that is at least partially hyperphosphorylated, mis-folded, post translationally modified, truncated, insoluble, present in an aggregated form, present in a tangle, or any combination thereof. A PATM can be characterized by an ability to bind non- physiologically active forms of the same macromolecule. A PATM can comprise a “disease state macromolecule” which generally refers to a form of a macromolecule the presence of which is associated with a disease.

[0065] The term “non-physiologically active target macromolecule” or “NPATM” generally refers to a target macromolecule that is not at least partially hyperphosphorylated, mis-folded, post translationally modified, truncated, insoluble, present in an aggregated form, present in a tangle, or any combination thereof. An NPATM can comprise a normal or healthy macromolecule. In some embodiments, a NPATM can bind to a PATM.

[0066] The term “physiologically active Tau protein” or “PAT protein” generally refers to a Tau protein, or a portion or fragment of a Tau protein, that is at least partially hyperphosphorylated, mis-folded, post translationally modified, truncated, insoluble, present in an aggregated form, present in a tangle, or any combination thereof.

[0067] The term “non-physiologically active tau protein”, “non-PAT protein”, or “normal Tau” generally refer to a Tau protein, or a portion or fragment of a Tau protein, that is not at least partially hyperphosphorylated, mis-folded, post translationally modified, truncated, insoluble, present in an aggregated form, present in a tangle, or any combination thereof.

[0068] The term “hyperphosphorylated Tau protein” or “HPT” can mean a Tau protein or a portion or fragment of a Tau protein that is phosphorylated at least at one position on the Tau protein. In some cases, HPT can mean a Tau protein or a portion or fragment of a Tau protein that is phosphorylated at residues 181, 199, 202, 205, 212, 214, 217, 231, 235, 262, 396, 404,422, or any combination thereof. In some cases, HPT can mean a Tau protein or a portion or fragment of a Tau protein that is phosphorylated at residues 217 and 231, or at one of residues 217 or 231, and also at another position on the Tau protein.

[0069] In some embodiments, “PAT-Tau” or “PAT-nTau” can refer to a complex of PAT with a non-physiologically active tau (non-PAT) protein (e.g., a normal Tau protein). In some embodiments, an AD biomarker can comprise PAT-Tau.

[0070] The terms “capture molecule”, “capture moiety”, “capture bead”, “capture particle”, and ’’target binding element”, as used interchangeably herein, generally refer to a molecule that is configured to capture a particular biomarker of interest (whether through direct or indirect binding to the biomarker) and that is bound (e.g., covalently or noncovalently, directly or through a linker, e.g., streptavidin-biotin or the like) to a solid support such as a particle (e.g., a microsphere, microbead, or the like). The capture moiety can bind directly to the biomarker and can be specific for that biomarker. The capture moiety can bind to one or more molecules that bind in turn to the biomarker to specifically capture it. Non-limiting examples of capture moiety comprise, but are not limited to, interference, polypeptides (e.g., antibodies, SH2 and other polypeptide binding domains, short synthetic peptides, and antigens), polynucleotides (e.g., polynucleotide capture probes, transcription factor binding sites, aptamers), oligonucleotides, antigens, biomarkers, polysaccharides, lipids, small molecules, molecular imprinted polymers (MIP), chimeric antibodies, therapeutic antibodies, recombinant antibodies, monoclonal antibodies, polyclonal antibodies, and antibody fragments thereof such as Fab, F(ab’)2, Fc, scFv, and engineered variants such as diabodies, triabodies, minibodies and single-domain antibodies. The capture moiety particles can be combined, mixed, or pooled to multiplex capture biomarkers using a plurality or pool of different biomarker capture particles (e.g., capture beads), each capture bead coated with a different target binding element or capture moiety, or beads coated with a mixture of different target binding elements or capture moieties, where all particles are magnetic, or where some particles are magnetic and others are non-magnetic.

[0071] The term “biomarker capture particle,” “target capture particle,” “analyte capture particle,” or “capture bead” as used herein, generally refers to particles comprising biomarker capture moieties that can interact with a biomarker in a sample.

[0072] The term “interference”, as used herein, generally refers to a substance present in, or a condition of, a sample, e.g., a biological sample, that can alter the correct value of the result by interfering with a capture moiety or a particle, or that can increase or decrease assay signal by bridging, steric hindrance, or autoantibody mechanisms. Erroneous results can occur unexpectedly with any specimen without the practical means to identify upfront such specimenslikely to cause problems. The consequence of such interference is that erroneous results can impact patient care, and can lead to unnecessary invasive, diagnostic, or therapeutic procedures, or failure to treat a patient with a false negative test result. Examples of interference include heterophile or heterophile-like interferences such autoantibodies, rheumatoid factor (RF), human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA) such as goat, rabbit, sheep, bovine, mouse, horse, pig, and donkey polyclonal and / or monoclonal antibodies, and manufacture assay-specific interference used in the test design or assay formulation such as the chemiluminescent substrate (isoluminol, luminol, ABEI, ruthenium, acridinium ester), fluorescent label (fluorescein or other fluorophores and dyes), anti-alkaline phosphatase (ALP), anti-horse radish peroxidase (HRP)), capture moieties (streptavidin, neutravidin, avidin, poly(A), poly DT, aptamers, antibodies, Fab, F(ab')2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers) and their binding partners (i.e. biotin, fluorescein, Poly(dT), Poly(A), antigen, etc.), conjugation linkers (LC, LC-LC, PEO, PEOn), anti-detection moiety interference (such as human anti-ALP, anti-ruthenium, anti-fluorescein, anti-ABEI interference), free biotin interference, and solid phase blocking proteins (bovine serum albumin, human serum albumin, ovalbumin, gelatin, purified poly- and monoclonal IgG such as mouse, goat, sheep and rabbit, polyvinyl alcohol or PAA, polyvinylpyrrolidone or PVP, Tween-20, Tween-80, Triton X-100, triblock copolymers such as Pluronic and Tetronic, and other commercially available blockers, blocking proteins and polymer-based blocking reagents such those from Surmodics and Scantibodies), lipids, triglycerides, bilirubin, hemolysis (hemoglobin, potassium), cholesterol, free biotin interference, anti-biotin interference, human antipolyethylene glycol (PEG) interference, anti-albumin, non-specific binding (NSB), anti-polymer antibodies, anti-polyvinylpyrrolidone (PVP) antibodies, anti-polyvinyl alcohol (PVA), autoantibodies, anti-conjugation linkers such as LC, LC-LC, and PEOn, over-the-counter (OTC) supplements, herbal remedies, or therapeutics that can cause problems that negatively impact test performance or accuracy, or cause erroneous results, of non-antibody based diagnostic tests such as molecular diagnostics or mass spectrometry (i.e. HPLC, MS, LCMS, LC-MS / MS), or antibody based tests such as radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), chemistry such as turbidimetric or particle-enhanced turbidimetric immunoassay (PETIA), lateral flow, flow cytometry, point-of-care (PoC), CLIA and CLIA waived tests and devices. Assay or analyte specific interference can comprise autoantibodies against an antibody or peptide used in the assay or test design, format, or protocol, or autoantibodies or immunoglobulins IgG, IgM, and / or IgA against the biomarker target such as macro complexes of endogenous Tau peptide andautoantibodies that accumulate in circulation or in the blood, serum, or plasma sample due to the poor renal clearance of large molecular weight protein macro complexes or poor clearance and accumulation due to renal insufficiency or disease (chronic kidney disease). More specifically, a Tau-based complex of autoantibodies against Tau, phosphorylated Tau, short Tau (truncated), long Tau (full length or N-terminal), linear peptide Tau, conformationally changed Tau, hyperphosphorylated Tau, or combinations thereof can accumulate in circulation and subsequently be detected in blood, serum or plasma as a falsely elevated level of Tau, phosphorylated Tau, short Tau (truncated), long Tau (full length or N-terminal), linear peptide Tau, conformationally changed Tau, hyperphosphorylated Tau, or combinations thereof by the test or assay which should only be detecting free or non-Macro complexes.

[0073] The term “interference capture particle” or “clean bead”, as used herein, generally refers to particles comprising capture moieties that will interact with a sample interference such that the interference, when exposed to the interference capture particle, will interact and bind to the particle surface. When the interference capture particles are isolated or removed from the sample via magnetic centrifugation, centrifugation, or filtration, the essentially particle-free sample will have a significantly lower level, concentration, threshold, or titer of the interference such that it will no longer interfere with a test or the measurement or characterization of the sample. Examples of capture moieties include human immunoglobulins (IgA, IgG, IgM, IgE) which may target autoantibody interference, animal antibodies (mouse, goat, sheep, rabbit, cow (bovine), lama, alpaca) which may target heterophilic antibodies such as HAMA, RF, and HAAA, polymerized antibodies or mouse antibody fragments (Fc, Fab, F(ab’)2) which may target HAMA and RF interference, proteins, enzymes, or small molecules (ALP, HRP, fluorescein / fluorophores, luminol, isoluminol, acridinium ester, ABEI, ruthenium, luciferin) that may target anti-signal generating interference, streptavidin, avidin, or neutravidin which may target biotin interference or biotin metabolite interference (bisnorbiotin, biotin sulfoxide), antibodies, aptamers, antibody fragments, MIPs, or polymers that may target specific interferences via epitope binding such as bilirubin, albumin, lipids, triglycerides, cholesterol, icterus (bile pigments), hemoglobin, herbal (for example, hemolysis, lipemia, icterus, tube additives, administration of radioactive or fluorescent compounds, drugs, herbal medicines, and nutritional supplements are all exogenous interferences that can adversely affect immunoassays), conjugation linkers such as LC, LC-LC, PEOn, PEG, anti-detection moiety interference (such as human anti-ALP, anti-ruthenium, anti-fluorescein, anti-ABEI interference), free biotin interference, the beads themselves as some sample interferences interact with the bead polymer or co-polymers, iron oxide or exposed iron crystals on the surface, PVP used as a wetting agentfor the polymerization, functional groups, or any NSB interactions with the bead. A sample can be pre-analytically cleaned of sample-specific interferences by adding interference capture particles to the sample, incubating the sample with the interference capture particles with or without mixing at ambient temperature, a cool temperature such as 2-8 degree Celsius, or a warm temperature such as 37 degree Celsius, and removing the interference capture particles from the sample using magnetic separation, magnetic isolation or removal, filtration, or centrifugation, prior to the detection, measurement, or quantitation of a biomarker, and a sample can be pre-analytically cleaned of non-specific binding (NSB) to the particle or solid phase using interference capture particles comprising the same or similar particle composition, or using the same lot of particles, as the “capture particles” whereby any bead-based or particle composition-specific interferences and NSB is captured by the interference capture particles prior to adding the target capture particles to the sample. Macro-Tau complexes and tau-specific or pTau-specific autoantibodies can be cleaned or removed from the sample using VeraBIND (Biomarker Isolation and N-richment for Detection) or clean beads coated with Tau peptide such as Tau441 or recombinant Tau peptides with or without phosphorylation (a pTaul81, a pTaul99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, a pTau422, or any combination thereof). Such autoantibody interference can be expected if a patient’s immune system detects brain derived tau in circulation as a foreign entity or antigen and generates an immune response or self-antibodies against said brain derived tau. The ability to selectively target and remove, reduce, or eliminate Macro-Tau complexes and autoantibodies against Tau can improve the accuracy and specificity of Tau-based or pTau-based tests or assays.

[0074] As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.

[0075] As used herein, the terms “treatment” or “treating” are used in reference to a administering to a subject a pharmaceutical or other intervention regimen for obtaining beneficial or desired results. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease cannot have been made.

[0076] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Introduction

[0077] Provided herein are methods and systems for detection and / or measurement of a biomarker derived from a sample from a subject. In some embodiments, the biomarker can comprise a binding of a physiologically active target macromolecule (PATM) to a non- physiologically active target macromolecule (NPATM). In some embodiments, a PATM can comprise a disease state macromolecule. In some embodiments, a NPATM can comprise a normal or non-disease state macromolecule. In some embodiments, a disease can comprise Alzheimer’s disease. In some embodiments, methods and systems disclosed herein can be used for early-stage screening, detection, and / or monitoring of a disease.Diseases caused by physiologically active macromolecule binding to non-physiologically active macromolecules

[0078] In some embodiments, the present disclosure provides a method of detecting a presence or absence of a biomarker for a disease in a subject. In some embodiments, a subject can be pre-symptomatic for a disease. In some embodiments, a subject can be suspected of having a disease but has not received a diagnosis. In some embodiments, the biomarker may comprise a physiologically active target macromolecule (PATM) or a portion or fragment thereof specific to the disease pathology. In some embodiments, the biomarker may comprise a conformation of the physiologically active target macromolecule or a portion or fragment thereof specific to disease pathology. In some embodiments, the biomarker may comprise a complex of the physiologically active target macromolecule or a portion or fragment thereof and a normal protein or a portion or fragment thereof (P ATM-normal protein complex). In some embodiments, the biomarker may comprise a conformation of the physiologically active target macromolecule or a portion or fragment thereof when the physiologically active target macromolecule or the portion or fragment thereof is in the PATM-normal protein complex. In some embodiments, the biomarker may comprise a binding of normal protein or a portion orfragment thereof to the PATM or a portion or fragment thereof. In some embodiments, the biomarker may comprise any conformation of the above.Creutzfeldt-Jakob disease

[0079] In some embodiments, the disease may comprise a Creutzfeldt-Jakob disease (CJD). In some embodiments, the PATM may comprise a physiologically active human prion protein (PrP). In some embodiments, the PATM may comprise a protein 14-3-3 in the cerebrospinal fluid (CSF). In some embodiments, the PATM may comprise CSF total tau protein. In some embodiments, the PATM may comprise a phosphorylated tau. In some embodiments, the PATM may comprise a CSF SlOOb protein. In some embodiments, a PrP antibody may be coated on a biomarker capture particle to capture a PrP protein or a portion or fragment thereof. In some embodiments, the PrP antibody may comprise a PrP monoclonal antibody. In some embodiments, an antibody of a physiologically active PrP may be coated on a biomarker capture particle to capture a physiologically active PrP protein or a portion or fragment thereof. In some embodiments, the antibody of the physiologically active PrP may comprise a PrP monoclonal antibody.

[0080] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to the PrP immunogen or a fragment thereof (e.g., an epitope of a PrP). In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of PrP with greater affinity than a NPATM form of PrP. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of PrP with lower affinity than a NPATM form of PrP. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of PrP with a similar affinity as a NPATM form of PrP. The PATM form of PrP may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of PrP (e.g., a non-disease state form of PrP or wild-type PrP). In some embodiments, the PATM form of PrP may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence ofMEHWGQPIPGAGQPWRQPLPTSGRWWLGAASWWWLGAASWWWLGAAPWWWLGT ASWWWLGSRRWHPQSVEQAE (SEQ ID NO: 35) or MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGG WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNM KHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRP MDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQ AYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 36). In some embodiments, the PATM form of PrP may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of PrP may comprise one or more aggregates of the NPATM form of PrP.

[0081] In some embodiments, the antibody molecule that binds to PrP (e.g., the PATM PrP) can be selected from the group consisting of: Prion protein Polyclonal Antibody (Catalog # TA590315), Prion protein PrP / CD230 Polyclonal Antibody (Catalog # BS-11788R), PrP Polyclonal Antibody (Catalog # PA5-96175), Prion protein PrP Polyclonal Antibody (Catalog # BS-4728R), PrP Polyclonal Antibody (Catalog # PA5-115797), PrP Polyclonal Antibody (Catalog # PA5-18539), PrP Polyclonal Antibody (Catalog # PA5-79878), PrP Polyclonal Antibody (Catalog # PA5-79877), PrP Polyclonal Antibody (Catalog # MA5-35724), and PrP Polyclonal Antibody (Catalog # Cat #MA5-49089). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of PrP.Amyotrophic Lateral Sclerosis

[0082] In some embodiments, the disease may comprise an amyotrophic lateral sclerosis (ALS) disease. In some embodiments, the PATM may comprise a physiologically active transactive response DNA binding protein 43 (TDP-43). In some embodiments, the PATM may comprise a phosphorylated TDP-43 protein. In some embodiments, a TDP-43 antibody may be coated on a biomarker capture particle to capture a TDP-43 protein or a portion or fragment thereof. In some embodiments, the TDP-43 antibody may comprise a TDP-43 recombinant rabbit monoclonal antibody. In some embodiments, an antibody of phosphorylated TDP-43 may be coated on a biomarker capture particle to capture a phosphorylated TDP-43 protein or a portion or fragment thereof. In some embodiments, the antibody of phosphorylated TDP-43 may comprise a phosphoro-TDP43 (Ser409, Ser410) polyclonal antibody.

[0083] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functionalfragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab’)2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to the TDP-43 immunogen or a fragment thereof (e.g., an epitope of a TDP-43). In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of TDP-43 with greater affinity than a NPATM form of TDP-43. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of TDP-43 with lower affinity than a NPATM form of TDP-43. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of TDP-43 with a similar affinity as a NPATM form of TDP-43. The PATM form of TDP-43 may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of TDP-43 (e.g., a nondisease state form of TDP-43 or wild-type TDP-43). In some embodiments, the PATM form of TDP-43 may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence of MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGI LHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTT EQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWC DCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFV TFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNS RGGGAGLGNNQGSNMGGGMNFGAF SINP AMMAAAQ AALQS S WGMMGMLAS QQNQ SGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSS MDSKSSGWGM (SEQ ID NO: 37). In some embodiments, the PATM form of TDP-43 may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of TDP-43 may comprise one or more aggregates of the NPATM form of TDP-43.

[0084] In some embodiments, the antibody molecule that binds to TDP-43 (e.g., the PATM TDP-43) can be selected from the group consisting of: TDP-43 Polyclonal Antibody (Catalog # PA5-20408), TDP-43 Recombinant Rabbit Monoclonal Antibody (23GB4730) (Catalog # MAS- 53798), TDP-43 Polyclonal Antibody (Catalog # PA5-89219), TARDBP Monoclonal Antibody (2E2-D3) (Catalog # Cat #H00023435-M01), TARDBP Monoclonal Antibody (SI) (Catalog # Cat #H00023435-M01J), TARDBP Monoclonal Antibody (1B4-B1) (Catalog # H00023435-M02), TDP-43 Monoclonal Antibody (GT733) (Catalog # MA5-27828), TDP-43 Polyclonal Antibody (Catalog # PAI-16996), TDP-43 Polyclonal Antibody (Catalog # PA5-20409), TDP- 43 Polyclonal Antibody (Catalog # PA5-19064), TDP-43 Polyclonal Antibody (Catalog # PAS- 19712), TDP-43 Monoclonal Antibody (6H6E12) (Catalog # 60019-2-IG), TDP-43 Monoclonal Antibody (1G4F5) (Catalog # 66734-1-IG), TDP-43 Monoclonal Antibody (1G5F6) (Catalog # 67345-1-IG), TDP-43 (C-terminal) Polyclonal Antibody (Catalog # 12892-1-AP), TDP-43 Polyclonal Antibody (Catalog # 18280-1 -AP), TARDBP TAR DNA binding protein / TDP43 Recombinant Rabbit Monoclonal Antibody (TARDP / 9299R) (Catalog # 23435-RBM3-P1), Phospho-TDP43 (Ser403, Ser404) Monoclonal Antibody (6B11B12) (Catalog # 66079-1-IG), Phospho-TDP43 (Ser409, Ser410) Monoclonal Antibody (1A2C1) (Catalog # 66318-1-IG), and Phospho-TDP43 (Ser409, Ser410) Polyclonal Antibody (Catalog # 22309-1-AP). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of TDP-43.Multiple Myeloma

[0085] In some embodiments, the disease may comprise a multiple myeloma (MM). In some embodiments, the PATM may comprise a Bence Jones protein. In some embodiments, the PATM may comprise an M protein. In some embodiments, the PATM may comprise free immunoglobulin light chains. In some embodiments, the PATM may comprise a haptoglobin. In some embodiments, the PATM may comprise a kininogen 1. In some embodiments, the PATM may comprise a transferrin. In some embodiments, the PATM may comprise a CD38. In some embodiments, the PATM may comprise a CD55. In some embodiments, the PATM may comprise a CD59. In some embodiments, the PATM may comprise a laminin receptor or fragment thereof. In some embodiments, an antibody of the MM PATM may be coated on a biomarker capture particle to capture an MM PATM or a portion or fragment thereof. In some embodiments, the antibody of the MM PATM may comprise a monoclonal antibody.

[0086] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab’)2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor (LR), or a functional fragment thereof (e.g., an epitope), or any combination thereof. In someembodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor with greater affinity than a NPATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor with lower affinity than a NPATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor with a similar affinity as a NPATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor. The PATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor (e.g., a non-disease state form or wild-type). In some embodiments, the PATM form of a laminin receptor immunogen may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence of MSGALDVLQMKEEDVLKFLAAGTHLGGTNLDFQMEQYIYKRKSDGIYIINLKRTWEKL LLAARAIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRFTPGTFTNQIQAAFREP RLLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWM LAREVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPA PEFTATQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTD WS (SEQ ID NO: 38). In some embodiments, the PATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of a Bence Jones protein, M protein, free immunoglobulinlight chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor may comprise one or more aggregates of the NPATM form of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor.

[0087] In some embodiments, the antibody molecule that binds to a target molecule of MM may comprise an antibody molecule that binds to PrP as described herein. In some embodiments, the antibody molecule that binds to a laminin receptor (e.g., the PATM laminin receptor) can be selected from the group consisting of: mouse LR monoclonal antibody (Catalog # MBS852692), Laminin Receptor / RPSA (Marker of Metastatic Potential) Recombinant Rabbit Monoclonal Antibody (RPSA / 6332R) (Catalog # 3921-RBM2-P0), Laminin Receptor / RPSA (Marker of Metastatic Potential) Recombinant Rabbit Monoclonal Antibody (RPSA / 6332R) (Catalog # 3921-RBM2-P1ABX), Laminin Receptor / RPSA (Marker of Metastatic Potential) Monoclonal Antibody (rRPSA / 6333) (Catalog # 3921-MSM3-P0), Laminin Receptor / RPSA (Marker of Metastatic Potential) Monoclonal Antibody (rRPSA / 6333) (Catalog # 3921-MSM3- P1ABX), 67kDa Laminin Receptor Monoclonal Antibody (OTI1G3), TrueMAB™ (Catalog # CF811423), Laminin Receptor / RPSA Monoclonal Antibody (RPSA, 2699) (Catalog # 3921- MSM1-P1), and Laminin Receptor / RPSA Monoclonal Antibody (RPSA, 2699) (Catalog # 3921-MSM1-P1ABX). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of a Bence Jones protein, M protein, free immunoglobulin light chain, haptoglobin, kininogen 1, transferrin, CD38, CD55, CD59, or a laminin receptor, or any combination thereof.Parkinson ’s Disease

[0088] In some embodiments, the disease may comprise Parkinson’s Disease. In some embodiments, the PATM may comprise an alpha-synuclein (a-syn). In some embodiments, the PATM may comprise a glial fibrillary acidic protein (GFAP). In some embodiments, the PATM may comprise beta-amyloid 1-42 (AB (1-42)). In some embodiments, the PATM may comprise a tau protein. In some embodiments, the PATM may comprise a phosphorylated tau protein. In some embodiments, the PATM may comprise a neurofilament light chain. In some embodiments, an antibody of the Parkinson Disease PATM may be coated on a biomarker capture particle to capture a PATM or a portion or fragment thereof. In some embodiments, the antibody of the Parkinson Disease PATM may comprise a monoclonal antibody.

[0089] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof cancomprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to the alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope), or any combination thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope) with greater affinity than a NPATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope). In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of alpha- synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope) with lower affinity than a NPATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope). In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope) with a similar affinity as a NPATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope). The PATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope) may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof (e.g., an epitope) (e.g., a non-disease state form or wild-type). In some embodiments, the PATM form of alpha-synuclein may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence of MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATV AEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGI LEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 39). In some embodiments, the PATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tauprotein, a neurofilament light chain, or a fragment thereof may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof may comprise one or more aggregates of the NPATM form of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof.

[0090] In some embodiments, the antibody molecule that binds to alpha-synuclein (e.g., the PATM alpha-synuclein) can be selected from the group consisting of: Phospho-a-Synuclein (Serl29) (D1R1R) Rabbit monoclonal antibody (Catalog # 23706), alpha Synuclein Monoclonal Antibody (Syn 505) (Catalog # 35-8300), alpha Synuclein Monoclonal Antibody (Syn 211) (Catalog # 32-8100), alpha Synuclein Monoclonal Antibody (Syn 211) (Catalog # AHB0261), alpha Synuclein Monoclonal Antibody (Syn 211) (Catalog # MA5-12272), Alpha-synuclein Monoclonal Antibody (LB509) (Catalog # 18-0215), alpha Synuclein Recombinant Rabbit Monoclonal Antibody (14H2L1) (Catalog # 701085), alpha Synuclein Polyclonal Antibody (Catalog # PA5- 17239), alpha Synuclein Polyclonal Antibody (Catalog # PAI -18264), alpha Synuclein Monoclonal Antibody (4B12) (Catalog # MAI -90346), a-Synuclein Monoclonal Antibody (1B10E9) (Catalog # 66412-1-IG), alpha-synuclein Polyclonal Antibody (Catalog # 10842-1-AP), alpha Synuclein Recombinant Rabbit Monoclonal Antibody (HL 1242) (Catalog # MA5-50306), and Phospho-alpha Synuclein (Serl29) Recombinant Rabbit Monoclonal Antibody (JB22-44) (MA5-34671). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of alpha-synuclein, GFAP, beta-amyloid 1-42, tau protein, phosphorylated tau protein, a neurofilament light chain, or a fragment thereof, or any combination thereof.Type II Diabetes

[0091] In some embodiments, the disease may comprise a Type II diabetes. In some embodiments, the PATM may comprise a follistatin protein. In some embodiments, the PATM may comprise an insulin-like growth factor binding protein 2. In some embodiments, the PATM may comprise a FABP4 protein. In some embodiments, the PATM may comprise a HGF protein. In some embodiments, the PATM may comprise a CTSD protein. In some embodiments, the PATM may comprise amylin (e.g., Islet Amyloid Polypeptide, IAPP). In some embodiments, an antibody of the Type II diabetes PATM may be coated on a biomarker capture particle to capture a Type II diabetes PATM or a portion or fragment thereof. In some embodiments, the antibody of the Type II diabetes PATM may comprise a monoclonal antibody.

[0092] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof (e.g., an epitope), or any combination thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof with greater affinity than a NPATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FAB4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof with lower affinity than a NPATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof with a similar affinity as a NPATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FAB4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof. The PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FAB4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FAB4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof (e.g., a non-disease state form or wild-type). In some embodiments, the PATM form of amylin (e.g., islet amyloid polypeptide) may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence ofMGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKRNAVEVLKREPLNYLPL (SEQ ID NO: 40) or KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 41) . In some embodiments, the PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof may comprise one or more aggregates of the NPATM form of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof.

[0093] In some embodiments, the antibody molecule that binds to amylin (e.g., islet amyloid polypeptide) can be selected from the group consisting of: Amylin Polyclonal Antibody (Catalog # PA5-84142), Amylin Polyclonal Antibody (Catalog # PA5-32261), Amylin Polyclonal Antibody (Catalog # PA5-98309), Amylin Monoclonal Antibody (C2) (Catalog # MA5-43963), Amylin Polyclonal Antibody (Catalog # PA5-102889), Amylin Polyclonal Antibody (Catalog # PA5-29713), Amylin Polyclonal Antibody (Catalog # 22305-1-AP), Amylin Peptide Polyclonal Antibody (Catalog # 500-3404), IAPP Polyclonal Antibody, MaxPab™ (Catalog # H00003375-D01P), and Amylin Polyclonal Antibody (Catalog # MUB0112S). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, a HGF protein, a CTSD protein, amylin, or a fragment thereof, or any combination thereof.Huntington ’s Disease

[0094] In some embodiments, the disease may comprise a Huntington's disease. In some embodiments, the PATM may comprise a glial fibrillary acidic protein. In some embodiments, the PATM may comprise beta-amyloid 1-42. In some embodiments, the PATM may comprise a tau protein. In some embodiments, the PATM may comprise a hyperphosphorylated tau protein. In some embodiments, the PATM may comprise a huntingtin (HTT) protein. In some embodiments, an antibody of the Huntington's disease PATM may be coated on a biomarker capture particle to capture a Huntington's disease PATM or a portion or fragment thereof. In some embodiments, the antibody of the Huntington's disease PATM may comprise a monoclonal antibody.

[0095] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functionalfragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof, or any combination thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a glial fibrillary acidic protein, betaamyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof with greater affinity than a NPATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof with lower affinity than a NPATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof with a similar affinity as a NPATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof. The PATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof (e.g., a non-disease state form or wild-type). In some embodiments, the PATM form of huntingtin protein (e.g., islet amyloid polypeptide) may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence ofMATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPPQA QPLLPQPQPPPPPPPPPPGPAVAEEPLHRPKKELSATKKDRVNHCLTICENIVAQSVRNSP EFQKLLGIAMELFLLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIKKNGAPRSLRAALWRFAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESVQETLAAAVPKIM ASFGNFANDNEIKVLLKAFIANLKSSSPTIRRTAAGSAVSICQHSRRTQYFYSWLLNVLL GLLVPVEDEHSTLLILGVLLTLRYLVPLLQQQVKDTSLKGSFGVTRKEMEVSPSAEQLV QVYELTLHHTQHQDHNVVTGALELLQQLFRTPPPELLQTLTAVGGIGQLTAAKEESGG RSRSGSIVELIAGGGSSCSPVLSRKQKGKVLLGEEEALEDDSESRSDVSSSALTASVKDEI SGELAASSGVSTPGSAGHDIITEQPRSQHTLQADSVDLASCDLTSSATDGDEEDILSHSSS QVSAVPSDPAMDLNDGTQASSPISDSSQTTTEGPDSAVTPSDSSEIVLDGTDNQYLGLQI GQPQDEDEEATGILPDEASEAFRNSSMALQQAHLLKNMSHCRQPSDSSVDKFVLRDEA TEPGDQENKPCRIKGDIGQSTDDDSAPLVHCVRLLSASFLLTGGKNVLVPDRDVRVSVK ALALSCVGAAVALHPESFFSKLYKVPLDTTEYPEEQYVSDILNYIDHGDPQVRGATAILC GTLICSILSRSRFHVGDWMGTIRTLTGNTFSLADCIPLLRKTLKDESSVTCKLACTAVRN CVMSLCSSSYSELGLQLIIDVLTLRNSSYWLVRTELLETLAEIDFRLVSFLEAKAENLHR GAHHYTGLLKLQERVLNNVVIHLLGDEDPRVRHVAAASLIRLVPKLFYKCDQGQADPV VAVARDQSSVYLKLLMHETQPPSHFSVSTITRIYRGYNLLPSITDVTMENNLSRVIAAVS HELITSTTRALTFGCCEALCLLSTAFPVCIWSLGWHCGVPPLSASDESRKSCTVGMATMI LTLLSSAWFPLDLSAHQDALILAGNLLAASAPKSLRSSWASEEEANPAATKQEEVWPAL GDRALVPMVEQLFSHLLKVINICAHVLDDVAPGPAIKAALPSLTNPPSLSPIRRKGKEKE PGEQASVPLSPKKGSEASAASRQSDTSGPVTTSKSSSLGSFYHLPSYLKLHDVLKATHAN YKVTLDLQNSTEKFGGFLRSALDVLSQILELATLQDIGKCVEEILGYLKSCFSREPMMAT VCVQQLLKTLFGTNLASQFDGLSSNPSKSQGRAQRLGSSSVRPGLYHYCFMAPYTHFTQ ALADASLRNMVQAEQENDTSGWFDVLQKVSTQLKTNLTSVTKNRADKNAIHNHIRLFE PLVIKALKQYTTTTCVQLQKQVLDLLAQLVQLRVNYCLLDSDQVFIGFVLKQFEYIEVG QFRESEAIIPNIFFFLVLLSYERYHSKQIIGIPKIIQLCDGIMASGRKAVTHAIPALQPIVHDL FVLRGTNKADAGKELETQKEVVVSMLLRLIQYHQVLEMFILVLQQCHKENEDKWKRL SRQIADIILPMLAKQQMHIDSHEALGVLNTLFEILAPSSLRPVDMLLRSMFVTPNTMASV STVQLWISGILAILRVLISQSTEDIVLSRIQELSFSPYLISCTVINRLRDGDSTSTLEEHSEGK QIKNLPEETFSRFLLQLVGILLEDIVTKQLKVEMSEQQHTFYCQELGTLLMCLIHIFKSGM FRRITAAATRLFRSDGCGGSFYTLDSLNLRARSMITTHPALVLLWCQILLLVNHTDYRW WAEVQQTPKRHSLSSTKLLSPQMSGEEEDSDLAAKLGMCNREIVRRGALILFCDYVCQ NLHDSEHLTWLIVNHIQDLISLSHEPPVQDFISAVHRNSAASGLFIQAIQSRCENLSTPTM LKKTLQCLEGIHLSQSGAVLTLYVDRLLCTPFRVLARMVDILACRRVEMLLAANLQSS MAQLPMEELNRIQEYLQSSGLAQRHQRLYSLLDRFRLSTMQDSLSPSPPVSSHPLDGDG HVSLETVSPDKDWYVHLVKSQCWTRSDSALLEGAELVNRIPAEDMNAFMMNSEFNLSLLAPCLSLGMSEISGGQKSALFEAAREVTLARVSGTVQQLPAVHHVFQPELPAEPAAYWSKLNDLFGDAALYQSLPTLARALAQYLVVVSKLPSHLHLPPEKEKDIVKFVVATLEALS WHLIHEQIPLSLDLQAGLDCCCLALQLPGLWSVVSSTEFVTHACSLIYCVHFILEAVAVQ PGEQLLSPERRTNTPKAISEEEEEVDPNTQNPKYITAACEMVAEMVESLQSVLALGHKR NSGVPAFLTPLLRNIIISLARLPLVNSYTRVPPLVWKLGWSPKPGGDFGTAFPEIPVEFLQ EKEVFKEFIYRINTLGWTSRTQFEETWATLLGVLVTQPLVMEQEESPPEEDTERTQINVL AVQAITSLVLSAMTVPVAGNPAVSCLEQQPRNKPLKALDTRFGRKLSIIRGIVEQEIQAM VSKRENIATHHLYQAWDPVPSLSPATTGALISHEKLLLQINPERELGSMSYKLGQVSIHS VWLGNSITPLREEEWDEEEEEEADAPAPSSPPTSPVNSRKHRAGVDIHSCSQFLLELYSR WILPSSSARRTPAILISEVVRSLLVVSDLFTERNQFELMYVTLTELRRVHPSEDEILAQYL VPATCKAAAVLGMDKAVAEPVSRLLESTLRSSHLPSRVGALHGVLYVLECDLLDDTAK QLIPVISDYLLSNLKGIAHCVNIHSQQHVLVMCATAFYLIENYPLDVGPEFSASIIQMCGV MLSGSEESTPSIIYHCALRGLERLLLSEQLSRLDAESLVKLSVDRVNVHSPHRAMAALGL MLTCMYTGKEKVSPGRTSDPNPAAPDSESVIVAMERVSVLFDRIRKGFPCEARVVARIL PQFLDDFFPPQDIMNKVIGEFLSNQQPYPQFMATVVYKVFQTLHSTGQSSMVRDWVML SLSNFTQRAPVAMATWSLSCFFVSASTSPWVAAILPHVISRMGKLEQVDVNLFCLVATD FYRHQIEEELDRRAFQSVLEVVAAPGSPYHRLLTCLRNVHKVTTC (SEQ ID NO: 42). In some embodiments, the PATM can comprise a fragment of a huntingtin protein comprising at least about 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, or greater than about 2000 amino acid residues. In some embodiments, the PATM can comprise a fragment of a huntingtin protein comprising at most about 2000, 1500, 1000, 500, 250, 200, 150, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or less than about 2 amino acid residues. In some embodiments, the PATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof may comprise one or more aggregates of the NPATM form of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof.

[0096] In some embodiments, the antibody molecule that binds to huntingtin protein can be selected from the group consisting of: huntingtin antibody (Catalog # NBP3-13106), Huntington Polyclonal Antibody (Catalog # PAI -003), Huntingtin Monoclonal Antibody (1HU-4C8) (Catalog # MA3-040), Huntingtin Monoclonal Antibody (HDB4E10) (Catalog # MA5-16703),Huntingtin Monoclonal Antibody (3-19) (Catalog # MAI -115), Huntingtin Monoclonal Antibody (4-13) (Catalog # MAI-058), Huntingtin Recombinant Rabbit Monoclonal Antibody (JB89-34) (Catalog # MA5-41256), Huntingtin Monoclonal Antibody (HDC8A4) (Catalog # MAI-82100), Huntingtin Polyclonal Antibody (Catalog # PA5-85721), Huntingtin Polyclonal Antibody (Catalog # PA5-17247), Phospho-Huntingtin (Ser421) Polyclonal Antibody (Catalog # PAI-087), Huntingtin Recombinant Rabbit Monoclonal Antibody (6F2) (Catalog # MAS- 49884), Huntingtin Recombinant Supercl onal Antibody (3HCLC) (Catalog # 710695), Huntingtin Polyclonal Antibody (Catalog # 720029), Huntingtin VHH-8His-Cys-tag Recombinant Alpaca Monoclonal Antibody (SAA1178) (Catalog # MA5-54602), and Huntingtin Polyclonal Antibody (Catalog # OSH00050W-100UL). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of a glial fibrillary acidic protein, beta-amyloid 1-42, a tau protein, a phosphorylated tau protein, a huntingtin protein (HTT), or functional fragment thereof, or any combination thereof.Cancer

[0097] In some embodiments, the disease may comprise a cancer. In some embodiments, the PATM may comprise a physiologically active p53 protein. In some embodiments, the PATM may comprise a mutant p53 protein. In some embodiments, a p53 antibody may be coated on a biomarker capture particle to capture a p53 protein or a portion or fragment thereof. In some embodiments, the p53 antibody may comprise a p53 monoclonal antibody. In some embodiments, an antibody of mutant p53 may be coated on a biomarker capture particle to capture a mutant p53 protein or a portion or fragment thereof. In some embodiments, the antibody of mutant p53 may comprise a monoclonal antibody of mutant p53.

[0098] In some embodiments, the disease may comprise a breast cancer. In some embodiments, the PATM may comprise a physiologically active p53 protein. In some embodiments, the PATM may comprise a mutant p53 protein. In some embodiments, a p53 antibody may be coated on a biomarker capture particle to capture a p53 protein or a portion or fragment thereof. In some embodiments, the p53 antibody may comprise a p53 monoclonal antibody. In some embodiments, an antibody of mutant p53 may be coated on a biomarker capture particle to capture a mutant p53 protein or a portion or fragment thereof. In some embodiments, the antibody of mutant p53 may comprise a monoclonal antibody of mutant p53.

[0099] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof cancomprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to a p53 protein, or functional fragment thereof, or any combination thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a p53 protein or functional fragment thereof with greater affinity than aNPATM form of a p53 protein or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a p53 protein or functional fragment thereof with lower affinity than a NPATM form of a p53 protein or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of a p53 protein or functional fragment thereof with a similar affinity as aNPATM form of a p53 protein or functional fragment thereof. The PATM form of a p53 protein or functional fragment thereof may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a NPATM form of a p53 protein or functional fragment thereof (e.g., a non-disease state form or wild-type). In some embodiments, the PATM form of huntingtin protein (e.g., islet amyloid polypeptide) may comprise an amino acid sequence with at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to a sequence ofMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPG PDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGT AKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRR CPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHY NYMCNS S CMGGMNRRPILTIITLEDS S GNLLGRNSFEVRVC ACPGRDRRTEEENLRKKG EPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDA QAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 43). In some embodiments, the PATM can comprise a fragment of a p53 protein comprising at least about 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, or greater than about 2000 amino acid residues. In some embodiments, the PATM can comprise a fragment of a p53 protein comprising at most about 2000, 1500, 1000, 500, 250, 200, 150, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or less than about 2 amino acid residues. In some embodiments, the PATM form of a p53 protein or functional fragment thereof may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or anycombination thereof). In some embodiments, the PATM form of a p53 protein or functional fragment thereof may comprise one or more aggregates of the NPATM form of a p53 protein or functional fragment thereof.

[0100] In some embodiments, the antibody molecule that binds to a p53 protein or functional fragment thereof can be selected from the group consisting of: p53 Monoclonal Antibody (DO-7) (Catalog # MA5-12557), p53 Monoclonal Antibody (DO-1) (Catalog # MAS- 12571), p53 Monoclonal Antibody (SP5) (Catalog # MA5-14516), p53 Monoclonal Antibody (DO-1) (Catalog # AHO0152), p53 Monoclonal Antibody (X77) (Catalog # MAI-12549), p53 Monoclonal Antibody (PAb 1801) (Catalog # 13-4000), p53 Monoclonal Antibody (BP53-12) (Catalog # MAI-19055), p53 Monoclonal Antibody (PAb 1801) (Catalog # MA5-11296), p53 Monoclonal Antibody (BP53-12), FITC (Catalog # MAI-19551), p53 Monoclonal Antibody (BD53-12) (Catalog # MAI-7629), p53 Polyclonal Antibody (Catalog # PA5-27822), Phospho- p53 (Serl5) Recombinant Rabbit Monoclonal Antibody (14H61L24) (Catalog # 700439), Phospho-p53 (Serl5) Monoclonal Antibody (C.381.0) (Catalog # MA5-15229), P53 Monoclonal Antibody (6C4B6) (Catalog # 60283-2 -IG), P53 Polyclonal Antibody (Catalog # 10442-1-AP), and Acetyl-p53 (Lys382) Recombinant Rabbit Monoclonal Antibody (10 H13L14) (Catalog # 701270). In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of a p53 protein or functional fragment thereof, or any combination thereof.Basal Cell Carcinoma

[0101] In some embodiments, the disease may comprise a basal cell carcinoma (BCC). In some embodiments, the PATM may comprise an asprosin protein. In some embodiments, the PATM may comprise a suprabasin protein. In some embodiments, the PATM may comprise a cyclooxygenase-2 (COX-2). In some embodiments, the PATM may comprise an ezrin protein. In some embodiments, an antibody of the BCC PATM may be coated on a biomarker capture particle to capture a BCC PATM or a portion or fragment thereof. In some embodiments, the antibody of the BCC PATM may comprise a monoclonal antibody.

[0102] In some embodiments, detection of the PATM (e.g., a physiologically active human prion protein) can comprise using an antigen binding domain, antibody molecule, or functional fragment thereof as described herein. The antibody molecule or functional fragment thereof can comprise a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. The antigen binding domain, antibody molecule, or functional fragment thereof may bind to an asprosin protein, a suprabasin protein, a cyclooxygenase-2(COX-2), an ezrin protein, or functional fragment thereof, or any combination thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof with greater affinity than aNPATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof with lower affinity than a NPATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof. In some embodiments, the antigen binding domain, antibody molecule, or functional fragment thereof may bind to a PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof with a similar affinity as a NPATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof. The PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof may comprise an amino acid sequence comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or greater than about 99% sequence identity to aNPATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof (e.g., anon-disease state form or wild-type). In some embodiments, the PATM can comprise a fragment of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX- 2), or an ezrin protein comprising at least about 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, or greater than about 2000 amino acid residues. In some embodiments, the PATM can comprise a fragment of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), or an ezrin protein comprising at most about 2000, 1500, 1000, 500, 250, 200, 150, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or less than about 2 amino acid residues. In some embodiments, the PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof may comprise a protein with one or more epigenetic modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, or any combination thereof). In some embodiments, the PATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof may comprise one or more aggregates of the NPATM form of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof.

[0103] In some embodiments, the antibody molecule that binds to a BMM PATM may comprise any p53 antibody molecule described herein. In some embodiments, the antibody molecules may be used in an assay method described herein for enhanced detection and / or capture of an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2), an ezrin protein, or functional fragment thereof, or any combination thereof.Alzheimer’s disease

[0104] Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by the progressive loss of neurons and synapses in the brain. AD is the most common cause of dementia, affecting over 50 million people worldwide. AD can be characterized by two major neuropathological hallmarks: amyloid plaques and tau tangles. Amyloid plaques are made up of a protein called beta-amyloid, while tau tangles are made up of abnormally hyperphosphorylated tau. AD has a prolonged pre-symptomatic phase during which changes in the brain, such as the accumulation of beta-amyloid plaques and tau tangles, begin. In some embodiments, patients who carry one or two copies of Apolipoprotein E4 (ApoE4) allele have a greater risk of developing AD than who do not carry ApoE4. The physiological changes produced by betaamyloid plaques, insoluble tau aggregates, and hyperphosphorylated tau neurofibrillary tangles in the brain can initiate years or even decades before noticeable cognitive symptoms manifest.

[0105] Early detection of AD or related tauopathies offers several benefits, including timely interventions potentially delaying or mitigating the cognitive decline associated with the disease and allowing patients to access clinical trials and supportive care services. Developing reliable diagnostic tests for early AD detection or related tauopathies is a significant research focus. While several successful diagnostic tests have been developed, they tend to be more effective in diagnosing symptomatic patients. There is a notable absence of minimally invasive screening tools for asymptomatic to early-stage AD detection or related tauopathies using easily accessible specimens.

[0106] Developing diagnostics for the pre-symptomatic stage of AD or related tauopathies is complex due to the subtle nature of early changes in the brain. Additionally, the variability in individual age-based, gender-based, and ethnicity-based responses to the disease further complicates the identification of reliable biomarkers. Pursuing effective and minimally invasive diagnostic tools for early-stage AD or related tauopathies is crucial to ongoing research. Early stages of AD or related tauopathies involve the presence of biomarkers at extremely low concentrations. Detecting these trace amounts of biomarkers requires highly sensitive detection methods which can also eliminate or minimize influence by other substances or interferences present in the sample.

[0107] To address the limitations of current diagnostic tests for AD, various alternative sample types can be used as substitutes for invasive procedures. While blood-based or plasma assays can serve as a secondary option for developing a diagnostic test, their adoption faces challenges such as: (a) quantitative assessment of AD biomarkers, (b) sample transport complexities with blood, plasma, or serum, and (c) unsupervised sample collection difficulties, especially in the target population. Alternative samples like dried blood spots (DBS) and saliva offer advantages in terms of ease of collection and preservation of native sample conditions with minimal additives. Recent studies have revealed differential expressions of various AD biomarkers in DBS and saliva, making them viable options to use as easy access specimens for AD screening tests.

[0108] The reliability of a screening test is dependent on the quality and integrity of the sample specimens, and the consistency in the collection, storage, transport, and reconstitution of these samples plays a pivotal role in determining the accuracy and dependability of any screening tool. To develop a reliable quantitative AD screening test, it is crucial to establish a methodology for collecting a fixed and consistent amount of plasma or saliva from both normal control individuals and AD patients.

[0109] There is a clinical need for a non-invasive and accessible blood-based, plasma-based, serum-based, saline oral rinse-based, or saliva-based screening test that detects biomarkers for AD, e.g., physiologically active Tau (PAT)-non-physiologically active Tau (non-PAT) binding in AD, or detects PAT bound to normal Tau, as a new AD biomarker for AD pathology and related tauopathies. Such a screening test would be able to accurately identify AD patients and distinguish them from healthy controls as PAT-normal Tau binding can be elevated in preclinical AD patients, even before they develop cognitive symptoms. This means a screening test based on PAT-normal Tau binding has the potential to revolutionize the diagnosis and management of AD and improve the lives of millions of people affected by AD, as it could be used to identify individuals at high risk of developing AD, detect AD in the early stages of the disease, and / or predict its progression allowing for early intervention and monitoring.

[0110] Provided herein in some aspects are methods and systems for detecting biomarkers for AD. The method can comprise a) providing a sample from a subject; and b) detecting a presence or absence of a biomarker for Alzheimer’s disease in the sample. In some embodiments, the biomarker can comprise: i) a protein specific to tau pathology and Alzheimer’s disease pathology; ii) a conformation of the protein specific to tau pathology and Alzheimer’s disease pathology; iii) a complex of the protein and a normal protein; iv) a conformation of the protein when the protein is in the complex; or v) a binding of the normalprotein to the protein specific to tau pathology and Alzheimer’s disease pathology; or vi) any combination of i) - v).

[0111] In some embodiments, the protein specific to tau pathology and Alzheimer’s disease pathology can be a hyperphosphorylated tau protein (HPT) or a physiologically active hyperphosphorylated tau (PAT) protein. In some embodiments, the normal protein can be a non-hyperphosphorylated tau protein.

[0112] Tau is a microtubule (MT)-associated protein that plays an important role in maintaining the structure and function of neurons. Tau consists of an N-terminus projection domain with two inserts (N1 and N2), a Proline Rich Region (PRR) subdivided into PRR1 and PRR2 which contains major tau phosphorylation sites, and an MT Binding Repeat domain (MTBR) composed of four imperfect repeat motifs (R1-R4) that participates in both MT binding and tau aggregation. The central nervous system (CNS) tau isoforms are distinguished by the presence of both (2N), one (IN), or neither (ON) of the N terminus inserts and the presence (4R) or absence (3R) of the second of the four MTBR repeats found in Tau 441, giving rise to a total of six isoforms: 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, and 0N3R. Under non-pathological conditions, tau protein associates with axonal microtubules and stabilizes the microtubule against depolymerization. PAT can comprise a phosphorylated protein containing up to 85 potential serine (S), threonine (T), and tyrosine (Y) phosphorylation sites. Tau plays a key role in neurodegenerative tauopathies and has been demonstrated to be prognostic or diagnostic for Alzheimer’s disease or dementia, or in the brain of a patient with Alzheimer’s disease (AD patient). Approximately 45 phosphorylation sites, including Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, and Ser422 have been identified to be predominantly in the proline-rich domain and the regions flanking the microtubule-binding domain. When phosphorylated tau proteins (pTau) are released from damaged neurons, their levels are elevated in the cerebral spinal fluid (CSF) and plasma of AD patients as compared to healthy controls. For example, phosphorylated Tau (pTau) at amino acids Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, or Ser422 also known as a pTaul81, a pTaul99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, and a pTau422, have emerged as clinically sensitive and specific CSF and blood-based AD biomarkers. While phosphorylation of Tau at one or more specific sites, such as Thrl81, Seri 99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, and Ser422 can provide a useful potential indication of AD, it cannot be used as a direct marker of AD Tau pathology. In AD Tau pathology, Tau can be hyperphosphorylated, which binds to normal Tauinstead of binding to tubulin, resulting in self-aggregation and the formation of Tau tangles. The R2 (VQIINK) and R3 (VQIVYK) sites are implicated in tau aggregation. In some embodiments, Tau tangles can be more directly responsible for the neuronal loss and cognitive decline that is seen in AD patients. In some embodiments, the binding of PAT to normal Tau can reflect the aggregation of Tau in AD patients. In some embodiments, the levels of PAT can be elevated in the blood of AD patients, even in the early stages of the disease. In some embodiments, the levels of PAT can be elevated in the blood of patients with other tauopathies, such as frontotemporal dementia (FTD) and / or progressive supranuclear palsy (PSP). In some embodiments, the binding of PAT-normal Tau or PAT bound to normal Tau can be used as an AD biomarker for AD pathology or related tauopathies.

[0113] In some embodiments, the PAT protein or a portion or fragment thereof can comprise Tau that is hyperphosphorylated, mis-folded, post translationally modified, insoluble, present in an aggregated form, present in a tangle, or any combination thereof. In some embodiments, the PAT comprises a microtubule-binding region (MTBR) of tau containing MTBR R1 residues 244-274, MTBR R2 residues 275-305, MTBR R3 residues 306-336, MTBR R4 residues, or combinations thereof; containing the MTBR sites implicated in tau aggregation comprising residues 275-280 (VQIINK) and 306-311 (VQIVYK); containing residues 225-242 near residue 243 and Rl; containing residues 226-264 near residue 260 within Rl; containing R1 residues 256-273 (VKSKIGSTENLKHQPGGG, SEQ ID NO: 29), R2 residues 287-304 (VQSKCGSKDNIKHVPPGG, SEQ ID NO: 30), R3 residues 318-335 (VTSKCGSLGNIHHKPGGG, SEQ ID NO: 31), R4 residues 350-364 (VQSKIGSLDNITHVPGGG, SEQ ID NO: 32), and combinations thereof; containing Rl residues 268-271 (HQPG), R2 residues 299-302 (HVPG), R3 residues 330-333 (HKPG), R4 residues 362-365 (HVPG), or combinations thereof. In some embodiments, the PAT protein or a portion or fragment thereof can comprise a tau protein that comprises an ability to bind non-PAT protein (e.g., normal Tau) or a portion or fragment thereof. In some embodiments, non-PAT protein or a portion or fragment thereof can comprise protein that is not associated with a disease state.

[0114] In some embodiments, the subject can be pre-symptomatic for Alzheimer’s disease. In some embodiments, the method can further comprise detecting a presence of Alzheimer’s disease pathology or a prognosis of developing Alzheimer’s disease in the subject when the biomarker for Alzheimer’s disease is present in the sample; or detecting an absence of Alzheimer’s disease pathology or a prognosis of not developing Alzheimer’s disease in the subject when the biomarker for Alzheimer’s disease is absent in the sample. In someembodiments, the detecting can comprise detecting the PAT-Tau complex by detecting a binding of a capture moiety that is specific to: a) the PAT-Tau complex, b) the conformation of PAT protein or a portion or fragment thereof in a PAT-Tau complex, c) the conformation of Tau in a PAT-Tau complex, or d) a combination of a), b), and / or c).

[0115] In some embodiments, the method provided herein can be a non-invasive and affordable way to screen for AD pathology and related tauopathies, making it possible to screen more people for the disease, including those who can be reluctant to undergo more invasive tests. In some embodiments, the method provided herein can help identify individuals who are at risk for developing AD or who are already in the early stages of the disease, allowing for earlier diagnosis and treatment, which could lead to better outcomes for patients. In some embodiments, the method provided herein can be used to monitor the progression of AD and the response to treatment, providing valuable information to clinicians and patients to help guide treatment decisions.

[0116] In some embodiments, the sample can comprise a first sample. In some embodiments, the method can further comprise monitoring the biomarker over time to determine an increase or decrease in an amount of the biomarker in a second sample of the subject, relative to the amount of biomarker in the first sample. In some embodiments, the method can comprise diagnosing the subject with Alzheimer’s disease based at least in part on detecting: i) the physiologically active Tau (PAT) protein specific to tau pathology and Alzheimer’s disease pathology; ii) the conformation of the PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer’s disease pathology; iii) the complex of PAT protein or a portion or fragment thereof and non-physiologically active tau (PAT-Tau complex); iv) the conformation of the PAT protein or a portion or fragment thereof when the PAT protein or a portion or fragment thereof is in the PAT-Tau complex; or v) the binding of non-PAT protein or a portion or fragment thereof to PAT protein or a portion or fragment thereof; or vi) any combination of i) - v). In some embodiments, the method can further comprise administering a treatment to the subject for Alzheimer’s disease. In some embodiments, the method can further comprise administering a treatment to the subject for Alzheimer’s disease. In some embodiments, the treatment can comprise a medicament or disease modifying therapy (DMT) that targets amyloid pathology (amyloid plaque), tau pathology, neurodegeneration, cognitive function, or any combination thereof. . In some embodiments, a treatment can comprise administering an anti-sense oligonucleotide (ASO) to a subject in need thereof. In some embodiments, a treatment can comprise administering a pharmaceutically effective amount of TRIM21 to a subject in need thereof. In some embodiments, the tau pathology comprises aninsoluble tau aggregate, a neurofibrillary tangle, a PAT protein or a fragment or portion thereof, a PAT-Tau complex, or any combination thereof. In some embodiments, the medicament can comprise a galantamine, a rivastigmine, a donepezil, or any combination thereof. In some embodiments, the method can further comprise determining a course of a disease progression based on the quantity of the biomarker detected, the quantity of the amount of tau that is physiologically active, or a combination thereof. In some embodiments, a signal to noise ratio of the detection can be more than about 3.0. In some embodiments, the medicament comprises a galantamine, a rivastigmine, a donepezil, or any combination thereof. In some embodiments, the medicament is a tau-directed therapy such as targeting tau PTMs (Tideglusib [NP031112, Nypta®, Zentylor™, GSK-3J3 inhibitor, NP12], Lithium, Sodium selenate [VEL015], LY3372689, and Salsalate), aggregation inhibitors (LMTM [TRxO237, LMT-X, Methylene Blue, TAI], ACI3024 [Tau MorphomerTM], and Curcumin [Diferuloylmethane, Longvida™]), tau expression inhibitors (BIIB080 [IONIS-MAPTRX, ISIS 814907]), microtubule stabilizers (Davunetide [NAP, AL-108] and Epothilone [BMS-241027]), active immunization (AADvacl [Axon peptide 108 conjugated to KLH] and ACI-35), passive immunization (RG7345 [RO6926496], Gosuranemab [BIIB092, BMS-986168, IPN007], Semorinemab [R07105705, MTAU9937A, RG6100], Zagotenemab [LY3303560], JNJ63733657, Bepranemab [UCB0107, UCB 0107, Antibody D], PNT001, BIIB076 [NI-105, 6C5huIgGl / l], Tilavonema [C2N8E12, ABBV8E12, HJ9.3], E2814, and Lu AF87908).

[0117] In some embodiments, the methods and compositions disclosed herein can selectively detect physiologically active Tau (PAT) (e.g., hyperphosphorylated Tau) binding to normal Tau, or selectively detect the complex or aggregate of PAT (e.g., hyperphosphorylated Tau) bound to normal Tau, in a biological fluid specimen (e.g., CSF, plasma, serum, whole blood, dried blood spot, dried plasma spot, saliva, saline oral rinse, urine, etc.) as a biomarker of Alzheimer’s pathology and related tauopathies. In some embodiments, the methods and compositions disclosed herein can be ultra-sensitive and ultra-specific. In some embodiments, the methods and compositions disclosed herein can detect early-stage biomarkers. In some embodiments, the methods and compositions disclosed herein can detect AD at a pre- symptomatic stage, enabling timely interventions.

[0118] In some embodiments, the method provided herein can detect multiple biomarkers simultaneously. AD is a complex disease with multiple biomarkers, and being able to capture and measure multiple biomarkers at once enhances diagnostic efficiency and provides a more comprehensive view of disease progression.

[0119] In some embodiments, the method provided herein can work with easy to collect andaccessible specimens. This aligns with the goal of enabling at-home sample collection, which can greatly facilitate routine screening.

[0120] In some embodiments, the method provided herein is compatible with different sample types, including plasma, DBS, saliva, and Saline oral rinse. This versatility allows for more widespread usage and can cater to individual preferences and situations.

[0121] In some embodiments, biomarkers that can indicate the early stages of AD can comprise proteins, e.g., beta-amyloid, phosphorylated tau, and / or ApoE4. These biomarkers can be found in cerebrospinal fluid (CSF) and can be detected in blood (e.g., plasma).

[0122] In some embodiments, the present disclosure discloses an assay method for selectively detecting hyperphosphorylated Tau binding to normal Tau, or selectively detecting the complex or aggregate of hyperphosphorylated Tau bound to normal Tau, in a biological fluid sample as a biomarker of Alzheimer’s pathology and related tauopathies.

[0123] In some embodiments, elevated pTau levels of a pTau!81, a pTau!99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, and / or a pTau422 in plasma are associated with AD pathology. Plasma can be collected for AD pTau testing by venous draw (e.g., phlebotomy), or finger prick (e.g., capillary, dried blood spots (DBS)), or capillary shoulder collection (e.g., RedDrop One, Tasso+, Tasso- M20, or equivalent).

[0124] In some embodiments, the method can comprise (i) pre-analytically conditioning and cleaning a sample and (ii) capturing physiologically active hyperphosphorylated Tau (or hyperphosphorylated Tau, used interchangeably herein) (PAT) or the PAT-normal Tau complex or aggregate. In some embodiments, (i) comprises using clean beads (e.g., interference capture particles) to capture a plurality of interferences, thereby to reduce, minimize, or eliminate sample-specific interference and non-specific binding below the assay blocking threshold, or to reduce, minimize, or eliminate autoantibodies against Tau or Macro-Tau antibody complexes. In some embodiments, (ii) comprises using a normal Tau coated solid phase to capture hyperphosphorylated Tau or using an anti-phosphorylated Tau coated solid phase to capture hyperphosphorylated Tau-normal Tau complex or aggregate.

[0125] In some embodiments, the capture of hyperphosphorylated Tau or hyperphosphorylated Tau-normal Tau complex or aggregate by the solid phase (or biomarker capture particles) can be enhanced by use of a specific binding buffer with a mildly alkaline pH (e.g., pH 7.5 to 8.5) comprising 100-200 mM sodium chloride and 0.05 to 0.10% Tween-20, and by incubating the solid phase with the sample at an elevated temperature, e.g., 37-40 degree Celsius.

[0126] In some embodiments, the method further comprises (iii) eluting captured hyperphosphorylated Tau or hyperphosphorylated Tau-normal Tau complex or aggregate. In some embodiments, the elution of captured hyperphosphorylated Tau from the normal Tau coated solid phase, or the elution of normal Tau from the captured hyperphosphorylated Tau- normal Tau complex from the anti-phosphorylated Tau coated solid phase, can be enhanced using an assay specific wash buffer with an acidic pH less than pH 7.0, a high salt such as 1000- 2000 mM sodium chloride, Triton X-100 greater than 0.2% (v / v), 0.05 to 0.010% Tween-20, and a reducing agent such as DTT or TCEP to cleave disulfide bonds instrumental in the initiation of Tau aggregation.

[0127] In some embodiments, the detection of hyperphosphorylated Tau captured and purified by the solid phase can use a capture moiety, such as a monoclonal antibody, specific to a sterically accessible amino acid sequence or epitope of the hyperphosphorylated Tau peptide such as an amino acid sequence comprising one or more of the phosphorylated amino acids Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, and Ser262, Ser396, Ser404, and Ser422.

[0128] In some embodiments, the detection of hyperphosphorylated Tau or normal Tau on the solid phase can use an anti- hyperphosphorylated Tau or anti-normal Tau antibody, binder, or capture moiety labeled or tagged with a detection moiety or signal generating moiety.

[0129] In some embodiments, the detection of a hyperphosphorylated Tau-normal Tau complex or aggregate captured and purified by the solid phase can use a conformation specific antibody generated against the complex or aggregate as the immunogen, but which does not bind to or cross-react to either hyperphosphorylated Tau or normal Tau alone.

[0130] In some embodiments, the detection of purified and eluted hyperphosphorylated Tau, or the detection of purified and eluted normal Tau, can use an antibody pair or binding pair where one or both binders have a signal detection moiety to generate signal and measure the level or concentration of the biomarker, or to generate signal in close proximity after the binding pair binds to the purified and eluted biomarker.

[0131] In some embodiments, the present disclosure provides an assay kit for carrying out the method. In some embodiments, the assay kit can be a sandwich immunoassay, an inhibition immunoassay, a proximity-based homogeneous immunoassay, or a combination thereof. In some embodiments, the present disclosure provides a composition comprising a monoclonal antibody that is specific to a PAT-Tau complex. In some embodiments, the present disclosure provides a composition comprising a monoclonal antibody that is specific to the conformation of PAT or a fragment or portion thereof in a PAT-Tau complex. In some embodiments, the presentdisclosure provides a composition comprising a monoclonal antibody that is specific to the conformation of Tau protein in a PAT-Tau complex. In some embodiments, the monoclonal antibody is a human monoclonal antibody or an animal antibody, or a humanized animal antibody. In some embodiments, the animal can comprise a mouse, a rabbit, or a sheep.Samples

[0132] In some embodiments, the sample can comprise any human or animal serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, semen or seminal fluid, cells, tissues, biopsy material, DNA, RNA, or any fluid, dissolved solid, processed solid material, oral fluids such as oral mucosal transudates (OMT), saliva (passive drool or swab), buccal samples (e.g., collected by swabbing or brushing upper or lower gums), saline oral rinse (SOR) or oral rinses, gingival crevicular fluid (GCF), sputum, sweat, tears, mucus, urine, stool (liquid and / or solid), vaginal fluid, milk, cerebrospinal fluid, peritoneal fluid, pleural fluid and digestive fluid. In some embodiments, the sample can comprise a sample from a human. In some embodiments, the sample can comprise a sample from an animal.

[0133] In some embodiments, the sample comprises a volume of about 10 pL to about 100 mL. In some embodiments, the volume of the sample can be about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, about 500 pL, about 550 pL, about 600 pL, about 650 pL, about 700 pL, about 750 pL, about 800 pL, about 850 pL, about 900 pL, about 950 pL, about 1 mL, about 1.5 mL, about 2 mL, about 2.5 mL, about 3 mL, about 3.5 mL, about 4 mL, about 4.5 mL, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 20 mL, about 40 mL, about 50 mL, about 75 mL, about 100 mL, about 150 mL, or about 200 mL, or a range defined by any of the aforementioned amounts. The sample volume can include at least 10 pL, at least 20 pL, at least 30 pL, at least 40 pL, at least 50 pL, at least 60 pL, at least 70 pL, at least 80 pL, at least 90 pL, at least 100 pL, at least 150 pL, at least 200 pL, at least 250 pL, at least 300 pL, at least 350 pL, at least 400 pL, at least 450 pL, at least 500 pL, at least 550 pL, at least 600 pL, at least 650 pL, at least 700 pL, at least 750 pL, at least 800 pL, at least 850 pL, at least 900 pL, at least 950 pL, at least 1 mL, at least 1.5 mL, at least 2 mL, at least 2.5 mL, at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, at least 5 mL, at least 5.5 mL, at least 6 mL, at least 6.5 mL, at least 7 mL, at least 7.5 mL, at least 8 mL, at least 8.5 mL, at least 9 mL, at least 9.5 mL, at least 10 mL, at least 20 mL, at least 40 mL, at least 50 mL, at least 75 mL, at least 100 mL, at least 150 mL, or at least 200 mL. In some embodiments, the sample volume is less than 10 pL, less than 20 pL, lessthan 30 pL, less than 40 pL, less than 50 pL, less than 60 pL, less than 70 pL, less than 80 pL, less than 90 pL, less than 100 pL, less than 150 pL, less than 200 pL, less than 250 pL, less than 300 pL, less than 350 pL, less than 400 pL, less than 450 pL, less than 500 pL, less than 550 pL, less than 600 pL, less than 650 pL, less than 700 pL, less than 750 pL, less than 800 pL, less than 850 pL, less than 900 pL, less than 950 pL, less than 1 mL, less than 1.5 mL, less than 2 mL, less than 2.5 mL, less than 3 mL, less than 3.5 mL, less than 4 mL, less than 4.5 mL, less than 5 mL, less than 5.5 mL, less than 6 mL, less than 6.5 mL, less than 7 mL, less than 7.5 mL, less than 8 mL, less than 8.5 mL, less than 9 mL, less than 9.5 mL, less than 10 mL, less than 20 mL, less than 40 mL, less than 50 mL, less than 75 mL, less than 100 mL, less than 150 mL, or less than 200 mL. In some embodiments, the sample can comprise a volume less than 200 pL. In some embodiments, the sample is a point-of-care (POC) finger prick collection sample, or a blood lancet that collects whole liquid blood samples (capillary shoulder collection device such as RedDrop One which is a virtually pain-free, low cost, easy-to-use, whole blood and plasma lancet and dispensing device for Home Health, Point-of-Care, Centralized, and Remote Clinical Trials testing and is a whole blood and plasma lancet device delivering whole blood and plasma), pediatric sample, geriatric sample, or IV drug user sample, where blood collection is challenging. In some embodiments, the sample can comprise a volume between about 200 pL and about 5 mL. In some embodiments, the sample can comprise a volume larger than about 5 mL where larger volumes can be needed for biomarker enrichment.

[0134] In some embodiments, the sample comprises a biofluid. In some embodiments, the biofluid comprises whole blood, plasma, serum, oral fluid (saliva, drool, oral mucosal transudates (OMT), or oral rinse e.g., saline oral rinse), mucus, urine, semen, vaginal fluid, milk, cerebrospinal fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid (CSF), tissues, sweat, tears, or digestive fluid.Saline Oral Rinse

[0135] Saline oral rinse (SOR) is a saliva-based sample type that can easily be collected at- home or at the clinic.

[0136] In some embodiments, the SOR sample can be collected 30 minutes after any food intake or drinks at any time of the day. In some embodiments, the SOR collection process can comprise a 30 second oral rinse with 5 mL of 0.9% NaCl in purified water followed by expectoration into a funnel and collection tube, or a simple and fast “Swish-N-Spif ’ saliva sample collection kit.

[0137] In some embodiments, the SOR collection process is highly effective and consistent in recovering biomarkers from the oral cavity, compared to standard saliva collection methods.

[0138] In some embodiments, the use of saline oral rinse is particularly beneficial for older patients with “dry mouth” conditions.

[0139] In some embodiments, the collected saliva sample can be diluted with 5 mL of saline, reducing viscosity and interference, thus improving subsequent test performance.

[0140] In some embodiments, SOR collection kits can include custom collection tubes constructed using Sarstedt’s proprietary low protein binding polypropylene resin, a technology recognized for its effectiveness in Alzheimer’s Dementia research to minimize AD biomarker adhesion or loss to the plastic surface during collection, storage, and shipping (transit).Capillary Collection

[0141] A new sample type that facilitates at-home self-collection or supervised collection by a health care practitioner is a capillary shoulder collection device such as the Tasso+ Device, Tasso+ Kit, and Tasso-M20 by Tasso Inc. The Tasso+ device is a blood lancet that collects whole liquid blood samples. Multiple standard collection tubes are compatible. The Tasso+ Kit includes a Tasso+ device, an IVD collection tube, and additional collection materials to provide user convenience such as alcohol swabs and bandages. The compatible tubes enable specific use depending on the tube utilized in the kit and its intended use. The Tasso-M20 device delivers whole dried blood samples from the patient to the lab. It can be used for PK (pharmacokinetic) monitoring in patients enrolled in clinical trials. Subjects can successfully collect volumetrically precise samples regardless of access to trial sites.

[0142] Another recently FDA 510(k) cleared capillary collection device for blood, serum, or plasma is RedDrop One by RedDrop Dx. RedDrop One is a virtually pain-free, low cost, easy- to-use, whole blood and plasma collection and dispensing system for Home Health, Point-of- Care, Centralized, and Remote Clinical Trials testing. RedDrop One is a whole blood and plasma collection device delivering whole blood and plasma.Patterned Dried Blood Spot (pDBS)

[0143] In some embodiments, another new sample type that can easily be collected at-home or at the clinic is patterned dried blood spot (pDBS).

[0144] In some embodiments, pDBS developed by the Bioanalytical and Materials Chemistry, at Tufts University, can effectively separates blood cells and platelets from plasma while ensuring a fixed amount of plasma is collected.

[0145] In some embodiments, the innovative design and special materials used to make their card allow the absorption of a fixed amount of plasma in each spot on the card (CV of 3%), rendering quantitative diagnostics based on dry blood samples possible.

[0146] In some embodiments, the levels of pTau217 and pTaul81 in capillary blood directly collected on a DBS card displayed a modest correlation when compared to liquid plasma samples added to the DBS card. In some embodiments, in contrast, the pTau217 and pTau!81 concentrations in plasma DBS exhibited a significantly stronger correlation with the venous DBS card, where a controlled volume of blood was spotted on the collection card25. In some embodiments, the data demonstrates the need for pDBS where a fixed amount of plasma is in each spot on the card and sample volume is not highly variable such as with the ADXIOO-Card from Advanced Dx Inc. and Whatman 903 Protein saver Card from Cytiva.

[0147] In some embodiments, plasma pTau biomarkers have the potential to be used as non- invasive and affordable tools for the diagnosis and monitoring of AD in a routine and / or large- scale clinical setting. In some embodiments, plasma AD tests are also relatively affordable and accessible to patients as compared to other AD diagnostic tests, such as amyloid PET scans. In some embodiments, they can be used to identify individuals at high risk of developing AD, even before they develop cognitive symptoms, allowing for early intervention and monitoring. In some embodiments, they can be used to pre-screen potential participants in clinical trials for AD treatments which can help enrich the study population with individuals who are most likely to benefit from the treatment. In some embodiments, they can be used to monitor the progression of AD in patients who have already been diagnosed to help to assess the effectiveness of treatment and identify patients who are at risk of rapid decline. pTau231

[0148] In some embodiments, phosphorylation of Tau at one or more specific sites, such as Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, and Ser422, can provide a useful potential indication of AD. In some embodiments, in AD Tau pathology, Tau becomes abnormally hyperphosphorylated (e.g., PAT) in a manner, which leads to its binding to normal Tau instead of its binding to tubulin, resulting in its selfaggregation and the formation of Tau tangles. In some embodiments, Tau tangles can be more directly responsible for the neuronal loss and cognitive decline that is seen in AD. In some embodiments, the binding of PAT to normal Tau reflects the aggregation of Tau in AD, and the levels of this biomarker can be elevated in the blood of people with AD, even in the early stages of the disease or in people with other tauopathies, such as frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP).

[0149] pTau231 is a blood-based biomarker. In some embodiments, pTau231 is a specific isoform of tau protein phosphorylated at the threonine 231 residue. In some embodiments, Tau is a microtubule-associated protein important for neuronal stability. In some embodiments, oneof the major neuropathological hallmarks of AD is hyperphosphorylation of the tau protein which leads to tau pathology and neuronal death. In some embodiments, pTau231 entangles normal functional tau protein causing non-functional tau protein aggregation. In some embodiments, plasma levels of pTau231 are elevated in AD and are correlated with amyloid- PET positivity. pTau217

[0150] In some embodiments, pTau217 is a highly specific and predictive indicator of AD pathology across all stages of the AD continuum where pTau231 is the earliest plasma biomarker of dementia of the Alzheimer’s type in older primary care patients when compared to plasma p-Tau 181, amyloid beta 42 / 40 (AP42 / AJ340), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP). In some embodiments, Plasma pTau217 is a biomarker of AB deposition in preclinical AD and appeared to be linked to both amyloid and tau pathology in symptomatic AD. In some embodiments, using pTau217 alone to select cognitively impaired participants for a clinical trial could significantly reduce costs, compared to selection by PET. In some embodiments, The PrecivityAD2® blood test simultaneously quantifies specific plasma amyloid beta and tau peptide concentrations to calculate the AJ342 / 40 ratio and ptau217 / nptau217 ratio. In some embodiments, these biomarkers are abundant in cerebrospinal fluid (CSF) and serve as key indicators of AD progression.Particles

[0151] In some embodiments, the particles can comprise microparticles. In some embodiments, the particles can comprise nanoparticles. In some embodiments, the particles comprise beads. In some embodiments, the particles comprise a metal. In some embodiments, the particles are magnetic.

[0152] In some embodiments, the particles described herein (e.g., microparticle, nanoparticle) can comprise a core or support. In some embodiments, the core or support can be a paramagnetic or superparamagnetic material which can experience a force in a magnetic field gradient, but do not become permanently magnetized. Non-limiting examples of paramagnetic or superparamagnetic material can comprise iron oxide, ferromagnetic iron oxide, Fe20s or FesO4, maghemite, or combinations thereof.

[0153] In some embodiments, the core or support can comprise ceramic, glass, latex, silica, metal, alloy, colloidal metal such as gold, silver or alloy, or polymers.

[0154] In some embodiments, the particles can comprise an organic polymer or copolymer. In some embodiments, the organic polymer or copolymer is hydrophobic. In some embodiments,the organic polymer or copolymer can comprise a material selected from the group consisting of, but not limited to polystyrene, derivatized polystyrene, poly(divinylbenzene), styrene-acylate copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene- oxyethylene), polymethyl methacrylate, polymethacrylate, polyurethane, polyglutaraldehyde, polyethylene imine, polyvinylpyrrolidone, polyvinyl alcohol, poly(A)crylic acid, N,N’- methylene bis-acrylamide, polyolefins, polyethylene, polypropylene, polyvinylchloride, poly(acrylonitrile), polysulfone, poly(ether sulfone), pyrolized materials, block copolymers, and copolymers of the foregoing, silicones, or silica, methylol melamine, a biodegradable polymer such as dextran or poly(ethylene glycol)-dextran (PEG-DEX), or combinations thereof.

[0155] In some embodiments, the surface of the particle can comprise functional groups or a plurality of functional groups for covalent attachment (coupling, conjugation or binding) of a binder, binding partner, capture moiety or combinations thereof such as carboxyl, tosyl, epoxy, amine, sulfhydryl, hydroxyl, ester, and maleimide, click chemistry functionality [Copper(I)- Catalyzed Azide- Alkyne Cycloaddition (CuAAC), Strain-promoted Azide- Alkyne Cycloaddition (SPAAC), Strain-promoted Alkyne-Nitrone Cycloaddition (SPANC), and Reactions of Strained Alkenes such as Alkene and Azide [3+2] cycloaddition, Alkene and Tetrazine inverse-demand Diels-Alder, and Alkene and Tetrazole photoclick reaction], hydrazone-based coupling functionality such as S-HyNic (succinimidyl-6-hydrazino- nicotinamide) and S-4FB (N-succinimidyl-4-formylbenzamide) heterobifunctional crosslinkers, and photoreactive chemistries.Interference Capture Particles

[0156] In some embodiments, the present disclosure provides an interference capture particle. In some embodiments, the interference capture particle can be configured to capture one or more interferences in a sample. In some embodiments, the one or more interferences, if not removed, can affect the accuracy and / or sensitivity of an assay method.

[0157] Immunoassay blockers (e.g., TRU Block Ultra™) can target all heterophile interferences. Immunoassay blockers can be used in diagnostic assays to reduce non-specific binding and other interference that can lead to false-positive results. In some embodiments, antibody interference from human anti-mouse antibodies (HAMA), rheumatoid factor (RH), and heterophilic antibodies (HA) are a major concern in both paired monoclonal sandwich assays and competitive assays. In IgM detection assays, IgG antibodies which are present in 10-15x higher concentrations than IgM can also reduce assay sensitivity due to their sheer abundance. Specialized blockers are required to reduce antibody interference from HAMA, HA, RF and IgG to ensure an assay's accuracy and improve its sensitivity.

[0158] In some embodiments, the interference capture particles (e.g., Clean Beads) disclosed herein can comprise a blocking moiety or a blocker. In some embodiments, the blocker can reduce non-specific binding and other interference that can lead to false-positive results. In some embodiments, the blocker can reduce aggregation of the interference capture particles. In some embodiments, the interference capture particles coated with the blocker can be mono-disperse. In some embodiments, the blocker can comprise a Blockmaster™ blocker. In some embodiments, the interference capture particles can comprise Trublock Clean Beads that comprise Blockmaster™ (CE210 and CE510) Quenched Streptavidin Base Beads (BM- Quenched SA Beads) coated with biotinylated TRU Block™ Ultra monoclonal antibodies. The BM-Quenched SA Beads can pre-analytically bind and significantly reduce, deplete or eliminate any anti-streptavidin, anti-biotin, and anti-PEG antibodies or capture moieties in a sample, as well as any sample-specific moieties that can non-specifically bind to the biomarker capture particles. The key to maximize HPT recovery with high assay sensitivity and reproducibility is to mitigate these different interference mechanisms prior to adding the biomarker capture particles to the conditioned and cleaned samples as these sample specific interference mechanisms can cause false low assay signal due (steric hinderance and decreased HPT capture efficiency or bead binding capacity) or false high assay signal (HAMA-like bridging mechanisms, bead aggregation or agglutination and poor bead wash efficiency or HPT capture efficiency).Biomarker Capture Particles

[0159] Disclosed herein in some embodiments are compositions comprising a biomarker capture particle. In some embodiments, the biomarker capture particles can comprise any suitable particles described herein or any combinations thereof.

[0160] In some embodiments, the biomarker capture particles can comprise a capture moiety. In some embodiments, the capture moiety can be coated on a surface of the biomarker capture particles. In some embodiments, the capture moiety can be covalently or non-covalently bound to the biomarker capture particles. The covalent binding can comprise any suitable binding chemistries, for example, attachment through one or more functional groups selected from the group consisting of carboxyl, hydroxyl, tosyl, epoxy, aldehyde, amine, amide, amino, hydrazide, isothiocyanate, maleimide, and sulfhydryl. In some embodiments, the capture moiety can be bound to the biomarker capture particles with an amine reactive reagent such as sulfo- NHS-LC-biotin.

[0161] In some embodiments, the capture moiety (i.e., antibody or antibody fragment such as SH-Fab) can be bound / coated to the biomarker capture particle by a cleavable bond. In someembodiments, the cleavable bond can be a disulfide bond (R-S-S-R). After washing or isolating the particles from the sample matrix, the particles can subsequently be treated with a solution containing a reducing agent such as TCEP or DTT to cleave the disulfide bond and release the capture moiety -biomarker complex into a solution for subsequent treatment or measurement.

[0162] In some embodiments, the capture moiety can capture one or more biomarkers from a sample. In some embodiments, at least a portion of the capture moiety can interact with at least a portion of the one or more biomarkers. In some embodiments, the capture moiety can comprise a protein, or a portion thereof, or a fragment thereof. In some embodiments, the capture moiety can comprise an anti-pTau protein, or a portion thereof, or a fragment thereof. In some embodiments, the pTau protein can comprise a pTau!81, a pTau!99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217, a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, a pTau422, or any combination thereof. In some embodiments, the capture moiety can comprise an anti-Thr!81, anti-Ser!99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti-Ser404, anti-Ser422 protein, or a portion thereof, or a fragment thereof, or a combination thereof.

[0163] In some embodiments, a biomarker capture particle can comprise an anti-pTau!81, anti-pTau!99, anti-pTau202, anti-pTau205, anti-pTau212, anti-pTau214, anti-pTau217, anti- pTau231, anti-pTau235, anti-pTau262, anti-pTau396, anti-pTau404 or anti-pTau422 Capture Bead, or pool or combination thereof.

[0164] In some embodiments, the biomarker capture particles may be coated with RD-073 (epitope human Tau between residue 159 and 163amino acids 1-20) antibody, ADx204 (epitope human Tau between residue 1 and 20) antibody, HT7 (epitope human Tau between residue 1 and 20) antibody, BT2 (epitope human Tau between residue 194 and 198) antibody, 77G7 antibody, or any combination thereof.

[0165] In some embodiments, a biomarker capture particle can comprise an anti-pTau217 Capture Bead. In some embodiments, the anti-pTau217 Capture Bead can comprise a biotinylated mouse monoclonal antibody against human phosphorylated threonine 217 (pTau217) coated on biomarker capture particles (e.g., Blockmaster Quenched Streptavidin Base Beads or BM-Quenched SA Beads). The anti-pTau217 Capture Beads can capture total pTau217 from the sample, including HPT phosphorylated at threonine 217.

[0166] In some embodiments, a biomarker capture particle can comprise an anti-pTau231 Capture Bead. In some embodiments, the anti-pTau231 Capture Bead can comprise biotinylated mouse monoclonal antibody against human phosphorylated threonine 231 (pTau231) coated onbiomarker capture particles (e.g., BM-Quenched SA Beads). The anti-pTau231 Capture Beads can capture total pTau231 from the sample, including HPT phosphorylated at threonine 231.

[0167] In some embodiments, the biomarker capture particles can comprise pooled (e.g., mixed) biomarker capture particles or pooled Capture Beads. In some embodiments, the biomarker capture particles can comprise pooled anti-pTau217 Capture Beads and anti-pTau231 Capture Beads. In some embodiments, the pooled anti-pTau217 Capture Beads and anti- pTau231 Capture Beads can capture total HPT phosphorylated at threonine 231, threonine 217, or both threonine 231 and threonine 217 from the sample. Phosphorylated threonine 217 sites can be significantly increased in Braak stage 0 prior to cognitive decline or cognitive inhibition. Phosphorylated threonine 231 sites can be significantly increased in the trans entorhinal region at Braak stage III / IV and show a progressive increase with increasing Braak stages. The use of a pool of both anti-pTau217 and anti-pTau231 antibody coated Capture Beads can improve the sensitivity of the assay for capturing, purifying, detecting, and characterizing the binding of total HPT to non-PAT regardless of the stage of AD or dementia. Other combinations or pools of antibody coated Capture Beads such as capture beads coated with antibodies against tau phosphorylation at sites Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404, and Ser422 could also be used to increase test sensitivity or to change test specificity for detecting tau pathology in cognitively normal (CN), mild cognitive inhibition (MCI), and / or dementia patients. In some embodiments, the assay may use a pool of antibody coated capture beads against pTau217 and pTau231, or SA-BM beads coated with anti-pTau217 antibody (clone RD-085 from ADx Neurosciences) and coated with anti-pTau231 antibody (clone ADx253 from ADx Neurosciences). In some embodiments, both antibodies may capture total HPT across the continuum of neurodegenerative tauopathy or from early stage disease (cognitively normal) to late state disease (MCI through dementia).

[0168] In some embodiments, the PAT protein or the portion or fragment thereof can comprise a pTau217. In some embodiments, the PAT protein or the portion or fragment thereof can comprise a pTau231. In some embodiments, the PAT protein or the portion or fragment thereof can comprise both a pTau217 and a pTau231.

[0169] In some embodiments, a pooled Capture Beads can comprise a first capture bead (e.g., Capture Bead 1) and a second capture bead (e.g., Capture Bead 2) with a ratio from 1:10 to 10:1. In some embodiments, the first capture bead may be coated or comprise a first capture moiety (e.g., a first antibody). In some embodiments, the second capture bead may be coated or comprise a second capture moiety (e.g., a second antibody). In some embodiments, the first capture moiety can bind with a first analyte. In some embodiments, the first capture moiety canbind with a second analyte. In some embodiments, a pooled Capture Beads can comprise a first capture bead (e.g., Capture Bead 1) and a second capture bead (e.g., Capture Bead 2) with a ratio of 1: 1. In some embodiments, the pooled capture beads can capture a plurality of analytes. In some embodiments, based on expected RFU ranges and minimum bead concentration of each capture bead, the concentration of a lower activity capture bead can be adjusted or increased to match the activity of the higher activity capture bead. In some embodiments, a pooled capture beads can comprise one or more capture beads comprising one or more different capture moieties. In some embodiments, the one or more different capture moieties may bind one or more different analytes.

[0170] In some embodiments, a mass of capture beads may be used. The mass of capture beads may be used per reaction. In some embodiments, a mass of individual capture beads (e.g., anti-pTau!81, anti-pTau!99, anti-pTau202, anti-pTau205, anti-pTau212, anti-pTau214, anti- pTau217, anti-pTau231, anti-pTau235, anti-pTau262, anti-pTau396, anti-pTau404 or anti- pTau422 capture beads) can be at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 60 pg, at least about 70 pg, at least about 80 pg, at least about 90 pg, at least about 100 pg, at least about 110 pg, at least about 120 pg, at least about 130 pg, at least about 140 pg, at least about 150 pg, or greater than about 150 pg per reaction. In some embodiments, a mass of individual capture beads (e.g., anti-pTaul81, anti-pTaul99, anti-pTau202, anti-pTau205, anti-pTau212, anti-pTau214, anti- pTau217, anti-pTau231, anti-pTau235, anti-pTau262, anti-pTau396, anti-pTau404 or anti- pTau422 capture beads) can be at most about 150 pg, at most about 140 pg, at most about 130 pg, at most about 120 pg, at most about 110 pg, at most about 100 pg, at most about 90 pg, at most about 80 pg, at most about 70 pg, at most about 60 pg, at most about 50 pg, at most about 45 pg, at most about 40 pg, at most about 35 pg, at most about 30 pg, at most about 25 pg, or less than about 25 pg per reaction. In some cases, a reaction may have a greater mass of individual capture beads if the beads are not pooled compared to a mass of individual capture beads in a reaction in which the beads are pooled. For example, there may be at least about 50 pg of capture beads (e.g., anti-pTaul81, anti-pTaul99, anti-pTau202, anti-pTau205, anti- pTau212, anti-pTau214, anti-pTau217, anti-pTau231, anti-pTau235, anti-pTau262, anti- pTau396, anti-pTau404 or anti-pTau422 capture beads) in a pooled reaction compared to at least about 100 pg of capture beads (e.g., anti-pTaul81, anti-pTaul99, anti-pTau202, anti-pTau205, anti-pTau212, anti-pTau214, anti-pTau217, anti-pTau231, anti-pTau235, anti-pTau262, anti- pTau396, anti-pTau404 or anti-pTau422 capture beads) in a not pooled reaction.

[0171] In some embodiments, the biomarker capture particles can further comprise a blocking moiety. In some embodiments, the biomarker capture particles can be further quenched with a biotin. In some embodiments, the biomarker capture particles can be further sonicated to ensure the biomarker capture particles are monodisperse and non-aggregated.

[0172] In some embodiments, after coating the Blockmaster Quenched Streptavidin Base Beads (BM-Quenched SA Beads) with biotinylated anti-pTau antibodies, and blocking the pooled Capture Beads with a Blocking Buffer (e.g., 1.0 % Casein, 10 mM Tris-HCl, 200 mM NaCl, 0.10% Tween® 20, 0.05% sodium azide, pH 7.6 ± 0.1) overnight at RT with mixing, the pooled Capture Beads can be quenched with biotin and sonicated to ensure the beads are monodisperse and non-aggregated. This quenching of any remaining or available streptavidin biotin binding sites with D-biotin can mitigate potential streptavidin bead aggregation and agglutination during bead storage via streptavidin affinity or binding to any free biotins linked to the biotinylated antibodies coated on the streptavidin capture beads. In some case, some of the biotinylated antibodies coated on the streptavidin beads can have two or more different biotins conjugated to the antibodies via a PEG linker such as PEO4, PEO8, PEO12, and PEO16, but only one of these linked biotins on the antibodies can sterically bind to the streptavidin (i.e., an antibody is 150 KDa in size and streptavidin is only in 52-26 KDa in size) on the beads while the other linked biotins can remain free and unconjugated and available to bind to another streptavidin on a different bead (bead aggregation) or interact or attract but not bind to another streptavidin on a different bead (agglutination). In some embodiments, biotin can ensure the streptavidin on the Capture Beads is quenched with biotin (all 4 binding sites occupied with biotinylated capture antibody or free biotin) to mitigate any possible streptavidin cross-reactivity or non-specific binding to an epitope on the Tau441-V5 peptide or to the V5 recombinant tag (false high result, increased NSB and assay background, decreased assay sensitivity and signal- to-noise / Limit of Detection / Limit of Quantitation) in the subsequent normal tau binding step in the assay protocol.

[0173] In some embodiments, the present disclosure provides a composition comprising a first population of particles and a second population of particles. In some embodiments, the composition may further comprise one or more populations of particles. In some embodiments, a particle of the first population of particles can comprise a first antibody that is specific to at least a portion of a first phosphorylated tau protein. In some embodiments, a particle of the second population of particles can comprise a second antibody that is specific to at least a portion of a second phosphorylated tau protein. In some embodiments, the first phosphorylated tau protein can be any of a pTau!81, a pTau!99, a pTau202, a pTau205, a pTau212, a pTau214, a pTau217,a pTau231, a pTau235, a pTau262, a pTau396, a pTau404, a pTau422, or any combination thereof. In some embodiments, the second phosphorylated tau protein can be any of pTau!81, pTau!99, pTau202, pTau205, pTau212, pTau214, pTau217, pTau231, pTau235, pTau262, pTau396, pTau404 or pTau422, or any combination thereof.

[0174] In some embodiments, the present disclosure provides a method, comprising contacting a sample comprising one or more phosphorylated tau proteins with a composition comprising a first population of particles and a second population of particles, as disclosed herein.Assay Method

[0175] In some embodiments, an assay method for detecting a biomarker for AD can comprise 1) pre-analytically clean a sample (e.g., CSF, plasma, serum, DBS, pDBS plasma, saliva, saline oral rinse, urine) with interference capture particles, 2) capture AD biomarkers from the cleaned sample with biomarker capture particles, 3) wash the biomarker capture particles to remove the sample matrix, and 4) elute and neutralize the purified and enriched AD biomarker for subsequent detection.

[0176] In some embodiments, interference capture particles can be added to the sample or biospecimen to pre-analytically capture and remove interference(s) from the sample to prepare an interference free or depleted sample (“Cleaned Sample”). In some embodiments, the interference can comprise a HAMA, RF, anti-mouse, anti-goat, anti-sheep, anti-rabbit, anti- bovine, anti-Streptavidin, anti-Biotin, anti-PEG, anti-Histidine or anti-polyhistidine tag (e.g., anti-6His Tag, anti-6His Tag), anti-PVP, non-specific binding or solid phase-specific immunoglobulin interference, or a combination thereof. In some embodiments, the interference may comprise anti-detection moiety interference (such as human anti-ALP, anti-ruthenium, antifluorescein, anti-ABEI interference. In some embodiments, the interference may comprise free biotin interference.

[0177] [JS2]Some assay formats are susceptible to free biotin interference such as Avidin / biotin based assay formats with delayed avidin capture of biotin-tagged capture or detection moieties.In some embodiments, the interference capture particles can be incubated with the biospecimen from 10-30 minutes at room temperature to 40 degrees Celsius, or preferably 30 min at 37 degrees Celsius with mixing. In some embodiments, after the sample incubation is complete, the interference capture particles can be removed or isolated from the Cleaned Sample using physical separation methods such as magnetic separation on a strong magnet or plate magnet, centrifugation such as 5-15 min at 1,500 x g or greater, or filtration using a 0.2 micron or larger porosity filter or filter plate.

[0178] Assay or analyte specific interference can comprise autoantibodies against an antibody or peptide used in the assay or test design, format, or protocol, or autoantibodies or immunoglobulins IgG, IgM, and / or IgA against the biomarker target such as macro complexes of endogenous Tau peptide and autoantibodies that accumulate in circulation or in the blood, serum, or plasma sample due to the poor renal clearance of large molecular weight protein macro complexes or poor clearance and accumulation due to renal insufficiency or disease (chronic kidney disease). More specifically, a Tau-based complex of autoantibodies against Tau, phosphorylated Tau, short Tau (truncated), long Tau (full length or N-terminal), linear peptide Tau, conformationally changed Tau, hyperphosphorylated Tau, or combinations thereof can accumulate in circulation and subsequently be detected in blood, serum or plasma as a falsely elevated level of Tau, phosphorylated Tau, short Tau (truncated), long Tau (full length or N- terminal), linear peptide Tau, conformationally changed Tau, hyperphosphorylated Tau, or combinations thereof by the test or assay which should only be detecting free or non-Macro complexes.

[0179] In some embodiments, use of interference capture particles, biomarker-specific capture beads, and capture bead washing provides the first degree of specificity and sensitivity, and the biomarker-specific Lumit™ immunoassay provides the second degree of specificity and sensitivity, for combined ultra-sensitive and ultra-specific detection and measurement of biomarkers from samples such as plasma, DBS, saliva, and saline oral rinse. This is a significant advancement in biomarker detection and offers the potential to identify early signs of AD such as the detection of hyperphosphorylated Tau binding to normal Tau, or detection of the hyperphosphorylated Tau-normal Tau complex.

[0180] FIG. 3 shows that the application of interference capture particles (or clean beads, used interchangeably herein) to a patient sample can lead to a significant reduction in sample interferences. 100 pL samples of human plasma contained 10 different immunoglobulin interferences: Anti-BSA, Anti-Bead, Anti-His Tag, Anti-PEG, Anti-Bovine IgG, Anti-Rabbit IgG, Anti-Sheep IgG, Anti-Mouse IgG, Anti-Goat IgG, and Anti-Streptavidin. The samples were treated with 0, 10, 20, and 50 pg of interference capture particles, respectively. The interference capture particles reduced sample interference signal of the 10 different immunoglobulin interferences by an average of about 73%. The interferences were reduced to a level of non-existence or non-critical to influence expected assay results. In some embodiments, a single interference or interferent can be removed from the sample in a single reaction. In some embodiments, multiple interferents can be removed from the sample in a single reaction. In some embodiments, the interference cleaning process can be repeated a few times.

[0181] In some embodiments, use of interference capture particles to clean the sample can substantially increase the signal-to-noise ratio (S / N) in a testing. Table 1 shows exemplary testing result of IgM RFU signal of serum samples in a Lyme disease serology IgM test, with or without cleaning with interference capture particles. When the serum sample was not treated with the interference capture particles, the test detected 4,932 problematic negative and 7,730 positive samples, having a S / N of 1.6. In contrast, when the serum sample was treated with the interference capture particles, the test detected 2,923 problematic negative and 11,506 positive samples, having a S / N of 3.9. The reduction in noise and concurrent amplification of the signal allows a negative sample to be distinguished from a positive one in a serology test with a significantly higher level of confidence. The selective removal of non-specific signals from complex samples can enhance assay sensitivity by eliminating steric hindrances from immunoglublin interferences, increasing biomarker yield and reducing background signal interference to achieve a higher signal-to-noise ratio.Table 1: IgM RFU signal with or without cleaning with interference capture particles

[0182] FIG. 4 shows the high-performance liquid chromatography (HPLC) purification of biotinylated monomeric IgG for clean and capture bead preparation. IgG as such or upon biotinylation can form aggregates that can interfere with antigen capture efficiency or introduce non-specific signals. The monomeric IgGs were selectively purified to prepare interference capture particles.

[0183] In some embodiments, the detecting can comprise detecting a PAT-Tau complex by detecting a binding of a capture moiety that is specific to: a) a PAT-Tau complex, b) a conformation of PAT protein or a portion or fragment thereof in a PAT-Tau complex, c) the conformation of Tau in a PAT-Tau complex, or d) a combination of a), b), and / or c). In some embodiments, a capture moiety that is specific to a PAT-Tau complex can comprise an antihyperphosphorylated antibody against a specific phosphorylation site, an antibody against a conformation of a PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer’s disease pathology, an antibody against a PAT-Tau complex, an antibody against a conformation of a PAT protein or a portion or fragment thereof when a PAT is in a PAT-Taucomplex, an antibody against the non-hyperphosphorylated Tau, or an anti-PAT polyclonal antibody. In some embodiments, the capture moiety can comprise a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, a Fab, a Fab’2, chimeric antibody, a chimeric Fab’2 antibody fragment, a molecular imprinted polymer (MIP), an aptamer, an alpaca nanobody or a llama derived nanobody. In some embodiments, the capture moiety can comprise two or more capture moieties. In some embodiments, a specific phosphorylation site can comprise Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404 or Ser422. In some embodiments, a non- hyperphosphorylated Tau can comprise recombinant human Tau-441or a fragment or portion of tau441 peptide such as full length peptide, truncated peptide, a C-terminal truncated normal tau peptide, or shorter normal tau peptides cleaved or fragmented at different reported sites (HPT (AD P-tau) has been shown to bind shorter truncated tau peptide Taul51-391 (HA-taul51-391 or HEK-293FT / taul51-391) expressed in HEK 293 cells and includes a HA tag at the C- terminus, much faster and stronger than the Tau-441 full length peptide), modified recombinant amino acid sequence in any position(s) in the peptide (for example, KHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLK-FITC which can represent a truncated normal tau peptide with the tau binding domains, residues or epitopes required for HPT binding to normal Tau to form insoluble tau aggregates and neurofibrillary tangles between MTBR R1 / R2 and MTBR R2 / R3), amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising the tau aggregation sequence at the begging of R2 (VQIINKtau aggregation site) and or R3 (VQIVYK tau aggregation site), or any combination thereof. In some embodiments, the detecting can be performed without cleaving or eluting the PAT-Tau complex or Tau from the biomarker capture particles. In some embodiments, the detecting can be performed after the PAT-Tau complex or Tau is eluted or cleaved from the biomarker capture particles.

[0184] In some embodiments, the detecting can comprise: capturing a PAT-Tau complex; releasing the PAT-Tau complex; and detecting the PAT-Tau complex. In some embodiments, the detecting can comprise; capturing a PAT-Tau complex; disrupting a PAT-Tau complex to release a non-hyperphosphorylated tau; and detecting the non-hyperphosphorylated tau.

[0185] In some embodiments, the detecting can comprise: capturing tau protein on a substrate; and determining whether a captured tau protein is PAT by exposing it to non-hyperphosphorylated tau, wherein non-hyperphosphorylated tau complexes with a captured tau when a captured tau protein is PAT. In some embodiments, a non-hyperphosphorylated tau can be labeled. In some embodiments, the label can comprise a fluorophore, a stable isotope, a mass tag, horse radish peroxidase (HRP), alkaline phosphatase (ALP), a Luciferase, a chemiluminescent substrate such as isoluminol, luminol, acridinium ester, or, ABEI, an HRP substrate such as TMB Substrate Solution, an enhanced HRP detection substrate such as FemtoMax™ Super Sensitive HRP Substrate by Rockland Immunochemicals, Chemiluminescent Substrate Solution (HRP Marker) by Aero Biosystems, QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit, 1-Step™ ABTS Substrate Solution, or SuperSignal™ West Femto Maximum Sensitivity Substrate by ThermoFisher, Novex™ HRP Chromogenic Substrate (TMB) or Novex™ ECL Chemiluminescent Substrate Reagent Kit by Invitrogen, or ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) substrate, CHMI Chemiluminescent Ultra Sensitive HRP Microwell Substrate, or LUMI Chemiluminescent Super Sensitive HRP Microwell and / or Membrane Substrate by Suromids, or Lumi-Phos HRP (PS- atto) by Lumigen, or other similar enhanced HRP substrates, an ALP chemiluminescence substrate such as Lumi-Phos 530, Lumigen APS-5 , or Lumi-Phos Plus by Lumigen, an ALP fluorescence substrate such as AttoPhos® Substrate by Promega, an electrochemiluminescence substrate such as ruthenium, a peptide such as Lumit® SmTrip9 or SmTriplO, oligonucleotide, a recombinant tag such as a polyhistidine tag (e.g., His-tag, an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. In some embodiments, a recombinant tag can comprise a hexa histidine-tag, 6xHis-tag, or His6 tag), a V5 tag (a small epitope tag GKPIPNPLLGLDST (SEQ ID NO: 23), which can be placed by molecular cloning at the N- or C-terminus of a protein of choice), a HA tag (YPYDVPDYA; SEQ ID NO: 24), a FLAG tag (DYKDDDDK; SEQ ID NO: 25), a c-Myc tag (EQKLISEEDL; SEQ ID NO: 26), a GSK tag (26 kDa sequence of 211 amino acids), an immune-PCR oligonucleotide labeled antibody or peptide, Tandem mass tagged antibodies or peptides, stable isotope labelled antibodies or peptides (e.g., peptides labeled with light isotopes (XH,12C,14N, and / or16O) are combined with peptides labeled with heavy isotopes (2H,13C,15N, and / or18O) prior to mass spectrometry (MS) and the relative peak intensity of the two, or a combination thereof. In some embodiments, a TMT can comprise a TMTzero, a non-isotopically substituted core structure; a TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; a TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; a 10-plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 pl ex versionwith a different reporter and mass normalizer than the original TMT, a TMTpro Zero, or any combination thereof.

[0186] In some embodiments, detection can be label free such as biolayer interferometry (BLI) and surface plasmon resonance (SPR) which are both label-free optical technologies that measure biomolecular interactions.

[0187] In some embodiments, the capture moiety can comprise a recombinant human Tau441 peptide. Human Tau441 peptide is a recombinant, lyophilized powder, expressed in HEK 293 cells, >95% (SDS-PAGE).

[0188] The V5 tag, Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr- (GKPIPNPLLGLDST; SEQ ID NO: 23), is a recombinant tag to provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. The V5 tag is a highly specific epitope of the recombinant Tau-441 peptide.

[0189] In some embodiments, the Tau441 peptide can comprise a poly-his tag (e.g., HHHHHHHH; SEQ ID NO: 27) and a V5 tag (e g., GKPIPNPLLGLDST; SEQ ID NO: 23) at the C-terminus. In some embodiments, the Tau441 peptide can have a sequence that is MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDE AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAK TPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD NIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSS TGSIDMVDSPQLATLADEVSASLAKQGLDRIRGRKLGPFEG ' / 7 / W / 7.7G777S7RTGHHH HHHHHGGQ (SEQ ID NO: 22), where the italic region denotes a V5 tag and the bolded region denotes a poly-his tag. In some embodiments, the capture moiety can comprise a fragment or portion of tau441 peptide such as full length peptide, truncated peptide, modified recombinant amino acid sequence in any position(s) in the peptide, amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising the tau aggregation sequence at the begging of R2 (VQIINKtau aggregation site) and or R3 (VQIVYK tau aggregation site), or any combination thereof. Insome embodiments, the capture moiety can comprise at least one of MTBR R1-R4. In some embodiments, the capture moiety can comprise a fragment or portion of tau441 peptide comprising MTBR R1-R4. In some embodiments, the capture moiety can comprise a sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the tau441 sequence. In some embodiments, the capture moiety can comprise a sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to a fragment or portion of tau441 peptide combining at least one of MTBR R1-R4.

[0190] Any recombinant tag can be used as an alternative to the V5 tag and the anti-V5 AP- conjugate antibody such as the 8x-His Tag, HHHHHHHH, of the recombinant Tau-441 and an anti-8x-His tag AP-conjugate antibody.

[0191] A C-terminal truncated normal tau peptide, or shorter normal tau peptides cleaved or fragmented at different reported sites, can also be used as an alternative to the full-length Tau- 441 peptide in the assay.

[0192] In some embodiments, HPT can bind shorter truncated tau peptide Taul51-391 (HA- taul51-391 or HEK-293FT / taul51-391) expressed in HEK 293 cells, with a HA tag at the C- terminus, much faster and stronger than the Tau-441 full length peptide. In some embodiments, truncation of Tau can selectively facilitate its pathological activities.

[0193] Alzheimer's disease brain contains tau fractions with differential prion-like activities. In some embodiments, HA-Taul51-391 and an anti-HA AP-antibody conjugate, or a similar truncated Tau peptide with a tag and corresponding anti-tag AP-antibody conjugate can be used for tau pathology assay instead of the Tau-441 -V5 and anti-V5 AP-antibody conjugate.

[0194] In some embodiments, a truncated normal tau peptide with the sequence FITC- KHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLK-FITC (SEQ ID NO: 33), which has tau binding domains, residues or epitopes required for HPT binding to normal Tau can be used to bind HPT to form insoluble tau aggregates and neurofibrillary tangles between MTBR R1 / R2 and MTBR R2 / R3. FITC at both the N-terminus and C-terminus is a way to detect this custom synthesized peptide itself via fluorescence detection. The FITC(s) can be replaced with a recombinant tag such as V5 for detection of this peptide via an anti-V5 tag conjugate antibody.

[0195] In some embodiments, the detecting can comprise detecting a conjugate or detection reagent in a sandwich immunoassay or an inhibition immunoassay. In some embodiments, a conjugate can comprise an antibody or capture moiety that directly or indirectly generates assay signal, fluorescence, light, bioluminescence, color, or a combination thereof. In some embodiments, a conjugate can comprise an HRP, an ALP, a Luciferase, a fluorophore, achemiluminescent substrate, an electrochemiluminescence substrate, a bioluminescent substrate, an immuno-PCR oligonucleotide labeled antibody or peptide, Tandem mass tagged antibodies or peptides (e.g., six varieties of TMT including TMTzero, a non-isotopically substituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; 10-plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 pl ex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero), stable isotope labelled antibodies or peptides (e.g., peptides labeled with light isotopes (JH,12C,14N, and / or16O) can be combined with peptides labeled with heavy isotopes (2H,13C,15N, and / or18O) prior to mass spectrometry (MS) and the relative peak intensity of the two forms provides an accurate indication of the relative levels of each peptide in the sample), or any combination thereof. In some embodiments, a chemiluminescent substrate can comprise an isoluminol, a luminol, an acridinium ester, an ABEI, or any combination thereof. In some embodiments, an electrochemiluminescence substrate can comprise ruthenium. In some embodiments, a bioluminescent substrate can comprise SmTrip9 or SmTriplO. In some embodiments, an anti- PAT capture moiety can be labeled with SmTrip9 and an anti-PAT-Tau capture moiety can be labeled with SmTriplO. In some embodiments, an anti -PAT capture moiety can be labeled with SmTriplO and an anti-PAT-Tau capture moiety can be labeled with SmTrip9.

[0196] In some embodiments, the detecting can comprise detecting a specific phosphorylated epitope of Tau. In some embodiments, a specific phosphorylated epitope of Tau can comprise anti-Thr!81, anti-Ser!99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti- Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti-Ser404, or anti-Ser422, and a conjugate can be against a different phosphorylated or non-phosphorylated Tau epitope. In some embodiments, the method can further comprise measuring an assay signal, dose, or PAT or PAT-Tau concentration. In some embodiments, the detecting of the biomarker can comprise detecting through electrophoresis, immunoblotting, immunoprecipitation, autoradiography, mass spectrometry, proteomics, protein separation, western blotting, protein identification, immunoassay, light scattering, spectrometry, calorimetry, or any combination thereof. In some embodiments, the detecting of the biomarker can comprise detecting through mass spectrometry (MS), time-of-flight mass spectrometry (TOF-MS), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), liquid chromatography- mass spectrometry (LC-MS), or LC- MS / MS, or biolayer interferometry (BLI), surface plasmon resonance (SPR), or other label-free optical techniques. In some embodiments, the peptide or protein being detected can be labeledwith light isotopes (JH,12C,14N, and / or16O), tandem mass tag (e.g., six varieties of TMT: TMTzero, a non-isotopically substituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; 10-plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 plex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero), heavy isotopes (2H,13C,15N, and / or18O), or any combination thereof. In some embodiments, the electrochemiluminescence substrate can comprise ruthenium. In some embodiments, the bioluminescent substrate can comprise SmTrip9 or SmTriplO. In some embodiments, the detecting of the biomarker can comprise binding the biomarker to biomarker capture particles comprising a biomarker capture moiety.

[0197] In some embodiments, the method may comprise separating or isolating a protein or a peptide by immunoprecipitation. In some embodiments, the method may comprise using mass spectrometry (MS) to determine a mass of a given peptide with and without a phosphorylation(s) to any amino acids in a given digested peptide mass. A peptide with a phosphorylation may have a different mass than a peptide without a phosphorylation. In some embodiments, the method may comprise calculating anon-PAT to PAT ratio (e.g., Tau217 / pTau217 ratio, Tau231 / pTau231 ratio, etc.) determined from MS. In some embodiments, the method may comprise calculating a normal Tau to pTau ratio (e.g., Tau217 / pTau217 ratio, Tau231 / pTau231 ratio, etc.) determined from MS. In some embodiments, the nTau to pTau ratio may better differentiate disease protein (AD or tauopathy) from non-disease protein (healthy controls).

[0198] In some embodiments, the method may comprise calculating and determining the mass ratio (e.g., by MS) of PAT in the sample to the recombinant Tau441 (tagged or untagged) that is added and captured by or bound to the immunoprecipitated / purified PAT. In some embodiments, the recombinant Tau441 can be a fragment or portion of tau441 peptide such as full length peptide, truncated peptide, modified recombinant amino acid sequence in any position(s) in the peptide, amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising the tau aggregation sequence at the begging of R2 (VQIINK tau aggregation site) and or R3 (VQIVYK tau aggregation site), or any combination thereof. In some embodiments, the method can comprise digesting both the PAT and non-PAT captured on the beads and analyzing for a ratio of PAT to non-PAT. In some embodiments, the method can comprise eluting and digestingboth the PAT and non-PAT captured on the beads and analyzing for a ratio of PAT to non-PAT. In some embodiments, detection by the MS method may better discriminate disease from nondisease. In some embodiments, detection by the MS method may improve test sensitivity (true positives. In some embodiments, detection by the MS method may improve test specificity (true negatives).

[0199] FIG. 5 shows a homogeneous, no-wash immunoassay that detects a given analyte in test samples. The immunoassay does not require the immobilization of detection antibodies to plates, beads, or other surfaces. The immunoassay uses a luminescent structural complementation system consisting of two small peptide tags and a reagent-based polypeptide (LgT). In the immunoassay, antibodies are chemically labeled with the peptide tags SmTrip9 and SmTriplO. When in the presence of a target analyte, the labeled antibodies bind the protein to bring the peptide subunits into close proximity, allowing the binding of LgT present in the solution to reassemble into a functional luminescent enzyme that generates a luminescent signal in the presence of furimazine substrate. To conduct the assay, the reagents can be added to a sample, followed by waiting up to 90 minutes or less, then detection reagent can be added and the assay can be read.

[0200] In some embodiments, normal Tau binding to purified PAT (e.g., immunoprecipitation using a Tau specific PAT capture antibody such as clones against total Tau such as clone RD-073 (amino acids 1-20), clone HT7 (aa 159-163), and clone BT2 (aa 194-198), or clones against the microtubule binding region (MTBR) such as MTBR-tau243 based monoclonal antibody clones HJ32.11 (aa 225-242 with proximity to aa 243) or HJ34.8 (aa 226- 264 with proximity to aa 260), or monoclonal antibody clone 77G7 against MTBR (the epitope mapping and sequence have been provided in FIGS. 12-14, e.g., aa residues 256-273 (VKSKIGSTENLKHQPGGG, SEQ ID NO: 29) or more specifically aa 268-271 (HQPG), aa residues 287-304 (VQSKCGSKDNIKHVPPGG, SEQ ID NO: 30) or more specifically aa 299- 302 (HVPG), aa residues 318-335 (VTSKCGSLGNIHHKPGGG, SEQ ID NO: 31) or more specifically aa 330-333 (HKPG), aa residues 350-364 (VQSKIGSLDNITHVPGGG, SEQ ID NO: 32) or more specifically aa 362-365 (HVPG)) can be detected by immune-PCR, mass spectrometry (MS), liquid chromatography-mass spectrometry (LC-MS), or LC-MS / MS. Once the PAT is capture to the antibody coated solid phase (or biomarker capture particles) via immunoprecipitation and washed, normal tau (or recombinant Tau with or without a tag) can be added to couple / bind to the PAT bound on the solid phase. After this binding interaction is compete, the solid phase can be washed, and the captured normal Tau can be belted and detected by MS whereby the normal tau or recombinant tau can be detected by MS via its mass signatureor profile, by labelling it and detecting the label (e.g., light isotopes, heavy isotopes, Tandem mass tag, or the recombinant tag itself such as His6 tag, FLAG, HA, V5, or c-Myc).

[0201] In some embodiments, the anti-Tau antibody coated (e.g., anti-total Tau, anti-Tau epitope, anti-phosphorylated Tau such as anti-Thrl81, anti-Serl99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti- Ser404, anti-Ser422, etc.), or capture moiety coated (e.g., polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2, normal Tau peptide or fragment thereof, phosphorylated Tau peptide or fragment thereof, molecular imprinted polymer or MIP, aptamer, alpaca or llama derived nanobody, Tau derived from Alzheimer’s brains, conformation-specific Tau, MTBR-specific Tau, etc.) biomarker capture particles can be added to the cleaned sample to capture hyperphosphorylated Tau (PAT), or hyperphosphorylated Tau-normal Tau complex or aggregate (PAT-Tau), in the absence of heterophilic interference and / or NSB to maximize total PAT or total PAT-Tau capture efficiency and recovery from the sample. In some embodiments, the biomarker capture particles comprises a coating with a single capture moiety, two or more capture moieties, a plurality of capture moieties co-coated on the particles, or as a pool of two or more different particles, against PAT, PAT-Tau, conformation-specific PAT, or phosphorylated Tau (pTau), such as an anti-pTau-181, 202, 205, 212, 214, 217, 231, 235, or 262 antibody, an anti-total Tau antibody such as the HT7 or BT2 antibodies from Fujirebio, Tau5, or the 11G1 antibody from Phanes Biotech, or the HJ32.11 and HJ34.8 antibodies from Washington University in St. Louis used in their CSF MTBR-tau243 test . In some embodiments, the biomarker capture particles can comprise a pool or 2 or more different antibody coated or capture moiety biomarker capture particles to maximize PAT capture and recovery from the sample.

[0202] FIG. 11 shows different amino acid epitopes of Tau that can be targeted by different antibodies, such as, antibody clone HT7 for amino acids (a.a.) 159-163, antibody clone BT2 for a.a. 194-198, antibody clone Tau5 for a.a. 210-230, and antibody clone 77G7 for a.a. 244-368. Antibody clone 77G7 can bind a repeating epitope that repeats 4 times in a.a. 268-271, a.a. 299- 302, a.a. 330-333, and a.a. 362-365. FIG. 12 shows the sequences of the binding epitopes for antibody 77G7. FIG. 13 shows a.a. sequence of 255-269 (MTBR1) and 286-300 (MTBR2) of the binding epitopes across the Tau441 peptide for antibody 77G7 with OD450 values. FIG. 14 shows a.a. sequence of 317-331 (MTBR3) and 349-363 (MTBR4) of the binding epitopes across the Tau441 peptide for antibody 77G7 with OD450 value.

[0203] In some embodiments, the antibody for capturing PAT can be 77G7 antibody. FIG. 20A shows a sequence of a light version of 77G7 (with 219 a.a.). FIG. 20B shows a sequenceof a heavy version of 77G7 (with 443 a.a.). FIG. 20C shows the CDR annotation with Chothia scheme for the light version of 77G7 shown in FIG. 20 A. FIG. 20D shows the CDR annotation with Chothia scheme for the light version of 77G7 shown in FIG. 20B.Table 10. Exemplary heavy chain and light chain CDRs and FWRs of tan- targeting binding domains.

[0204] In some embodiments, the present disclosure provides an antibody molecule or antigen binding fragment thereof. In some embodiments, the antigen binding fragment thereof comprises a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody. An antibody for targeting tau can comprise a heavy chain variable region (VH) and a light chain variable region (VL). The antibody can comprise a binding domain that binds to an antigen (e.g., tau), epitope, or any combination thereof. The antibody can comprise any complementarity determining region (CDR) amino acid sequence, framework region (FR) amino acid sequence, or variable region amino acid sequence described herein. In some embodiments, an antibody can comprise any complementarity determining region (CDR) amino acid sequence, framework region (FR) amino acid sequence, or any combination thereof as set forth in Table 10.Table 11. Exemplary variable regions and CDRs of tau- targeting binding domains

[0205] In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a heavy chain variable region (VH) comprising a sequence having at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 10. Insome embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a heavy chain variable region (VH) comprising a sequence having at most about 99.5%, at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 94%, at most about 93%, at most about 92%, at most about 91%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a heavy chain variable region (VH) comprising a sequence having between about 20% to about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a heavy chain variable region (VH) comprising a sequence having between about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 10.

[0206] In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a light chain variable region (VL) comprising a sequence having at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a light chain variable region (VL) comprising a sequence having at most about 99.5%, at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 94%, at most about 93%, at most about 92%, at most about 91%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a light chain variable region (VL) comprising a sequence having between about 20% to about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a light chain variable region (VL) comprising a sequence having between about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 11.

[0207] An amino acid sequence for any CDR, heavy chain variable region, and / or light chain variable region can comprise an amino acid sequence as set forth in Table 11. In some embodiments, an antibody described herein (e.g., a tau-targeting antibody molecule) can comprise a CDR-H1 as set forth in SEQ ID NO: 1, 4, or 6, a CDR-H2 as set forth in SEQ ID NO: 2 or 5, and a CDR-H2 as set forth in SEQ ID NO: 3; and / or a CDR-L1 as set forth in SEQ ID NO: 7, a CDR-L2 as set forth in SEQ ID NO: 8, and a CDR-L3 as set forth in SEQ ID NO: 9. The antibody can be used in an assay as described herein.

[0208] In some embodiments, the biomarker capture particles can be incubated with the biospecimen from 10-30 minutes at room temperature, or more preferably a minimum of 30 min at 37 degree C, with mixing. In some embodiments, after the sample incubation is complete, the biomarker capture particles can be isolated from the cleaned sample using magnetic separation. In some embodiments, the magnetic bead-based process can facilitate using larger sample volumes such as 0.2 mL, 1 mL, 5 mL, 10 mL, or greater than 10 mL of sample such as dried blood spot or dried plasma spot reconstitution / elution buffer sample, 0.5-2.0 mL neat saliva, drool, or saliva with preservation agent, or 1-5 mL saline oral rinse, preferably 1.0-1.3 mL SOR, with or without preservation agent (i.e. anti-microbial, anti-viral, anti-fungal, anti-protease, antiphosphatase, anti-enzymatic activity, anti-mucin, and / or anti-secretory agglutinins, or a mixture thereof of 2 or more of these sample preservation agents), to capture and enrich low abundance AD biomarkers such as PAT or PAT-Tau from larger sample volumes.

[0209] In some embodiments, the capture antibody can be generated against PAT purified from an AD brain and used as the immunogen assuming it has a different conformation or tertiary structure or shape, or different epitopes, as compared to pTau or PAT purified from normal or non- AD brains. In some embodiments, a conformation-specific antibody can be used to detect PAT from AD patients. In some embodiments, the antibody can be generated against purified PAT or PAT-Tau complex from AD brain, or against an immunogen made by binding purified PAT from AD brain with normal Tau peptide or recombinant peptide e.g., from PhanesBiotech, assuming such monoclonal antibodies generated against such as immunogen will not detect or cross-react with pTau, PAT, or PAT-Tau from healthy controls or non- AD diseased patients.

[0210] In some embodiments, after the PAT or PAT-Tau is captured by biomarker capture particles or solid phase, the particles or solid phase surface can be washed 1 to 3 times using a Wash Buffer to remove the biospecimen sample matrix and other sample derived constituents from the particles, and / or to wash the surface of the particles to remove any non-specifically bound or absorbed sample constituents not specifically captured and bound by the antibodies or capture moieties prior to eluting the captured total PAT from the biomarker capture particles. In some embodiments, after the PAT or PAT-Tau is captured by biomarker capture particles or solid phase, the particles or solid phase surface can be washed, e.g., 2 times using a Wash Buffer to remove the biospecimen sample matrix and other sample derived constituents from the Capture beads, and / or to wash the surface of the Capture beads to remove any non-specifically bound or absorbed sample constituents not specifically captured and bound by the antibodies or capture moieties prior to eluting the captured total PAT from the biomarker capture particles. In some embodiments, the wash buffer can comprise a detergent (0.05-0.10% Tween-20), salts (150-200 mM NaCl, 10-20 mM TRIS), and pH 7.0-8.0 to improve bead mixing and bead monodispersity during resuspensions with the Wash Buffer while disrupting non-affmity or nonantibody based hydrophobic and ionic binding interactions on the surface of bead surface. In some embodiments, the mixed and resuspended biomarker capture particles in the Wash Buffer can be incubated to improve wash efficiency and to maximize biomarker recovery by the capture beads or solid phase. In some embodiments, the mixed and resuspended biomarker capture particles in the Wash Buffer can be incubated to recapture any PAT or PAT-Tau that can have been initially dissociated from the biomarker capture particles during the addition of the Wash Buffer, to maximize biomarker recovery by the capture beads or solid phase. In some embodiments, the mixed and resuspended biomarker capture particles in the Wash Buffer can be incubated, or incubated with mixing, preferably incubated 1-5 min at 37 degrees Celsius with mixing, to improve wash efficiency, but also to recapture any PAT or PAT-Tau that can have been initially dissociated from the biomarker capture particles during the addition of the Wash Buffer, to maximize biomarker recovery by the capture beads or solid phase.

[0211] In as aspect, the present disclosure provides an assay method for detecting a biomarker in a sample. The biomarker is associated with a disease and is different from a normal biomarker (non-diseased). The assay method can comprise pre-conditioning the sample with a pre-conditioning composition. The assay method can comprise removing one or moreinterferences from the sample. In some embodiments, removing the one or more interferences can comprise contacting the sample with a plurality of interference capture particles. The assay method can further comprise capture the biomarker from the sample. In some embodiments, capturing the biomarker can comprise contacting the sample with a plurality of biomarker capture particles, thereby forming a plurality of biomarker-particle complexes. In some embodiments, the plurality of biomarker capture particles can comprise pooled biomarker capture particles. In some embodiments, the pooled biomarker capture particles can comprise two or more types of biomarker capture particles, wherein the two or more types of biomarker capture particles can comprise different capture moieties. The assay method can further comprise contacting the plurality of biomarker-particle complexes with a plurality of normal biomarker, thereby forming a plurality of normal biomarker-biomarker-particle complexes. The assay method can further comprise contacting the plurality of normal biomarker-biomarker- particle complexes with a plurality of conjugates, thereby forming a plurality of conjugatenormal biomarker-biomarker-particle complexes. In some embodiments, the plurality of conjugates can comprise one or more detection moieties. In some embodiments, the one or more detection moieties can be detected by a suitable detection method. In some embodiments, the one or more detection moieties can directly or indirectly generate an assay signal, e.g., fluorescence, HRP fluorescence, ALP fluorescence, light, bioluminescence, immune-PCT signal, color, or mass signature, or a combination thereof. In some embodiments, the conjugate can comprise an HRP, an ALP, a Luciferase, a fluorophore, a chemiluminescent substrate, an electrochemiluminescence (ruthenium), or a bioluminescent substrate, or any combination thereof. In some embodiments, a presence of the one or more detection moieties can indicate a presence of the conjugate-normal biomarker-biomarker-particle complexes, and further indicate a presence of the biomarker in the sample. In some embodiments, an absence of the one or more detection moieties can indicate an absence of the conjugate-normal biomarker-biomarker- particle complexes, and further indicate an absence of the biomarker in the sample.

[0212] In as aspect, the present disclosure provides a method for detecting a biomarker in a sample. The method can comprise contacting the sample with a composition comprising a first reagent and a second reagent. In some embodiments, the composition can comprise a preconditioning composition or buffer as disclosed in the present application. In some embodiments, the first reagent can deactivate or reduce the activity of one or more interference enzymes. In some embodiments, the one or more interference enzymes can comprise one or more endogenous phosphatases. In some embodiments, the one or more interference enzymes can degrade or deactivate the biomarker. In some embodiments, the one or more endogenousphosphatases can de-phosphorylate a physiologically active protein (e.g., an HPT). In some embodiments, the second reagent can solubilize the biomarker. In some embodiments, the method can further comprise contacting the sample with a plurality of biomarker capture particles, wherein the plurality of biomarker capture particles can comprise a capture moiety to bind the biomarker. In some embodiments, the method can further comprise, prior to contacting the sample with the plurality of biomarker capture particles, contacting the sample with a plurality of interference capture particles, wherein the plurality of interference capture particles can bind one or more interference molecules in the sample. In some embodiments, the second reagent can further reduce non-specific binding of the biomarker to the plurality of interference capture particles. In some embodiments, the composition can further comprise a third reagent. In some embodiments, the third reagent can block one or more biotin binding moieties. In some embodiments, the third reagent can comprise biotin.Protocol

[0213] The VeraBIND Tau assay can be a novel blood test of tau pathology. Unlike traditional methods that focus on quantifying biomarker levels, the VeraBIND Tau test can identify the presence of active tau pathology, which can provide a more direct measure of tau- related disease. VeraBIND Tau can be an in vitro bead-based ELISA that uses chemiluminescence to measure the binding of hyperphosphorylated tau (HPT) to normal tau, indicating active tau pathology, which can be a hallmark of AD.

[0214] A technical benefit of the VeraBIND Tau assay can be its detection of pathologically functional HPT captured and purified from a non-invasive blood-based EDTA plasma sample. This detection of physiological activity (e.g., the prion-like seeding of non-PAT to HPT) can be unique and functionally more relevant for detecting AD pathology compared to other bloodbased biomarker tests, which primarily quantify specific forms of phosphorylated tau. Other tests can capture and / or quantify insoluble tau aggregates from invasive cerebrospinal fluid (CSF) and plasma samples. These other tests provide a quantitative assay that can be incapable of assessing the physiological activity of the captured Tau aggregates. In comparison, the level or amount of non-PAT binding to HPT detected in the VeraBIND Tau assay can agree against tau PET scans which correlate with the progression or stage of AD and the level of HPT-non- PAT interaction. The assays described herein can therefore provide enhanced methodologies for detection of a biomarker representative of AD progression (e.g., tau and / or hyperphosphorylated tau) and results of the assay can correspond to progression of AD as seen in neuroimaging scans (e.g., tau PET scans).

[0215] In some embodiments, the method provided herein (e.g., VeraBIND sampletransformation and biomarker purification technology protocol) can comprise:1) conditioning two different 2.0 mL Deep Well Plates #1 and #2 by filling each well of both plates with VeraBIND Wash Buffer A, soak for 5 min, and then aspirate and discard Wash Buffer A;2) adding 100 pL interference capture particles for Antigen Testing to the conditioned 2.0 mL Deep Well Plate #1;3) adding sample [(100 pL plasma + 1,200 pL PBS), or (DBS eluate, or pDBS plasma eluate, diluted up to 1,300 pL with PBS), or 500 pL saliva diluted up to 1,300 pL with PBS, or 1,300 pL SOR] to the 100 pL of interference capture particles in each well of Plate #1 and incubating for 30 min at 37 °C with mixing on the Qinstruments Heater / Shaker at 1,000 rpm with thermal adaptor for the 2.0 mL Deep Well plates;4) magnetically separating the interference capture particles in Plate #1 on the Alpaqua 96-well plate magnets to i) first pull the interference capture particles down to the bottom of each well in Plate #1 using the Alpaqua Magnum FLX magnet for 10 min, and ii) then to separate the interference capture particles to the side of the wells in Plate #1 with the slotted Alpaqua Catalyst 96 magnet for 4 min;5) adding 100 pL of anti-PAT or anti-PAT-Tau antibody coated biomarker capture particles to the corresponding wells of the conditioned 2.0 mL Deep Well Plate #2;6) aspirating 1,300 pL of cleaned sample from each well in Plate #1 and transferring to corresponding wells in Plate #2, and incubating for 30 min at 37 °C with mixing on the Qinstruments Heater / Shaker at 1,000 rpm with thermal adaptor for the 2.0 mL Deep Well plates;7) magnetically separating the biomarker capture particles in Plate #2 on the Alpaqua plate magnets to i) first pull the biomarker capture particles down to the bottom of each well in Plate #2 using the Magnum FLX magnet for 10 min, and ii) then to separate the biomarker capture particles to the side of the wells of Plate #2 with the slotted Catalyst 96 magnet for 4 min;8) aspirating and discarding the -1,400 pL sample supernatant (i.e., removing the sample matrix);9) washing the biomarker capture particles 2X in Plate #2 using 1.0 mL of VeraBIND Wash Buffer B (i.e., TBS, 0.05% Tween-20). For each wash, 1 mL of VeraBIND Wash Buffer B is added to the biomarker capture particles and incubated for 1.5 min at 37 °C with mixing on the Qinstruments Heater / Shaker at 1,000 rpm with thermal adaptor for the 2.0 mL Deep Well plates. After each incubation, the biomarker capture particles are magnetically separated to the bottom of each well in Plate #2 on the Magnum FLX magnet for 5 min,followed by the Catalyst 96 magnet for 2 min, to pellet the biomarker capture particles, and aspirate and discard the wash buffer;10) adding 180 pL of VeraBIND Elution Buffer to the biomarker capture particles in Plate #2 and incubating for 1.5 min at 37 °C with mixing on the Qinstruments Heater / Shaker at 1,800 rpm with thermal adaptor for the 2.0 mL Deep Well plates;11) magnetically separating the biomarker capture particles in Plate #2 on the Alpaqua Catalyst 96 magnet for 4 min to pellet the biomarker capture particles;12) adding 26.6 pL of VeraBIND Neutralization Buffer in each corresponding well of a 96-well standard volume Black clear bottom Plate #3 (low binding resin, black, flat clear bottom 96-well plate, Coming Part Number 3631);13) aspiratingl74 pL of sample eluate from Plate #2 and transferring to corresponding wells in Plate #3; and14) testing the total PAT or PAT-Tau. The total PAT or PAT-Tau is purified into 200 pL of VeraBIND buffer sample pH 7.2 (TRIS-based buffer, 0.05-0.10% Tween-20, pH 7.7), and ready for testing by immunoassay (e.g., Immunoassay, ELISA, or Lumit®).Assay Format #1

[0216] In some embodiments, after washing the Biomarker capture particles or solid phase, the PAT or PAT-Tau captured by the Biomarker capture particles or solid phase can be directly measured by use of an anti-phosphorylated Tau (anti-Thr!81, anti-Ser!99, anti-Ser202, anti- Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti- Ser396, anti-Ser404, anti-Ser422, or any combination thereof), anti-PAT (or more specifically an epitope, phosphorylation site, or epitope unique to PAT and not present in normal phosphorylated Tau), anti-PAT-Tau (the complex of PAT bound to normal Tau), anticonformation-specific PAT, anti-conformation specific PAT-Tau, or anti-normal Tau (the normal Tau bound to the PAT-Tau complex, or more specifically an epitope unique to normal Tau not present in phosphorylated Tau or PAT) conjugate or detection reagent for a sandwich immunoassay or inhibition immunoassay. In some embodiments, the conjugate can comprise an antibody or capture moiety that directly or indirectly generates assay signal, fluorescence, HRP fluorescence, ALP fluorescence, light, bioluminescence, immune-PCT signal, color, mass signature, or a combination thereof. In some embodiments, the conjugate can comprise an HRP, an ALP, a Luciferase, a fluorophore, a chemiluminescent substrate, an electrochemiluminescence (ruthenium), or a bioluminescent substrate, or any combination thereof. In some embodiments, the conjugate can comprise Lumit® (SmTrip9 or SmTriplO if the anti-PAT or anti-PAT-Tau capture moiety on the capture beads or solid phase is also labeledwith the alternate SmTrip9 or SmTrip 10 (i.e., SmTrip9 conjugate and SmTriplO solid phase), or if the normal Tau on the capture beads or solid phase is also labeled with the alternate SmTrip9 or SmTriplO). In some embodiments, if the VeraBIND capture moiety detects pTau231 or another specific phosphorylated epitope of Tau such as such anti-Thrl81, anti-Serl99, anti- Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti- Ser262, anti-Ser396, anti-Ser404, or anti-Ser422, then conjugate can be against a different phosphorylated or non-phosphorylated Tau epitope (FIG. 13). In some embodiments, for a sandwich immunoassay, assay signal, dose, or PAT or PAT-Tau concentration will be directly proportional to the amount of PAT or PAT-Tau captured on the capture beads or solid phase and subsequently detected by the conjugate and remaining after washing excess or non-bound conjugate away from the capture beads or solid phase, or the more PAT or PAT-Tau captured on the capture beads or solid phase the more anti-PAT or anti-PAT-Tau conjugate can bind and assay signal will increase or be higher.

[0217] In some embodiments, the chemiluminescent substrate can comprise an isoluminol, a luminol, an acridinium ester, an ABEI, a HRP substrate, an ALP substrate an immune-PCR oligonucleotide labeled antibody or peptide, stable isotope labelled antibodies or peptides (e.g., peptides labeled with light isotopes (XH,12C,14N, and / or16O), peptides labeled with heavy isotopes (2H,13C,15N, and / or18O), tandem mass tagged antibodies or peptides (e.g., six varieties of TMT: TMTzero, a non-isotopically substituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; 10-plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 plex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero), or any combination thereof. In some embodiments, peptides labeled with light isotopes (1H,12C,14N, and / or16O) can be combined with peptides labeled with heavy isotopes (2H,13C,15N, and / or18O) prior to mass spectrometry (MS) and the relative peak intensity of the two forms can provide an accurate indication of the relative levels of each peptide in the sample. In some embodiments, the electrochemiluminescence substrate can comprise ruthenium. In some embodiments, the bioluminescent substrate can comprise SmTrip9 or SmTriplO. In some embodiments, an anti-PAT capture moiety can be labeled with SmTrip9 and an anti-PAT-Tau capture moiety can be labeled with SmTriplO. In some embodiments, an anti-PAT capture moiety can be labeled with SmTriplO and an anti-PAT-Tau capture moiety can be labeled with SmTrip9. In some embodiments, the detecting of the biomarker can comprise detecting a specific phosphorylated epitope of Tau. In some embodiments, the specificphosphorylated epitope of Tau can comprise anti-Thrl81, anti-Serl99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti- Ser404, or anti-Ser422, and the conjugate can be against a different phosphorylated or nonphosphorylated Tau epitope.

[0218] In some embodiments, the Lumit™ proximity -based homogeneous immunoassay the capture beads or solid phase does not need to be washed after adding the SmTrip-labeled conjugate as signal will only be generated if this conjugate binds to the PAT or PAT-Tau captured by the Lumit-labelled capture moiety on the capture beads or solid phase.

[0219] In some embodiments, for an inhibition immunoassay, assay signal, dose, or PAT or PAT-Tau concentration will be inversely proportional to the amount of PAT or PAT-Tau captured on the Tau-coated capture beads or solid phase, and subsequently the amount of conjugate that binds and remains on the capture beads or solid phase after washing excess conjugate from the capture beads or solid phase. In some embodiments, if normal Tau coated capture beads or solid phase has captured PAT or PAT-Tau then the anti-Tau conjugate binding will be impaired, sterically hindered, or reduced as the conjugate will not be able to freely bind to the Tau on capture beads or solid phase. In some embodiments, in the absence of PAT or PAT-Tau, or with lower concentrations of PAT or PAT-Tau in the sample, more conjugate, or maximum conjugate, will bind for increased assay signal. In some embodiments, a high assay signal correlates to a low dose or absence of PAT or PAT-Tau. In some embodiments, a low assay signal correlates to a high dose or presence of PAT or PAT-Tau. In In some embodiments, the Lumit™ proximity-based homogeneous immunoassay capture beads or solid phase does not need to be washed after adding the SmTrip-labeled conjugate. In some embodiments, an assay signal will only be generated if the conjugate binds to the SmTrip-labelled normal Tau on the capture beads or solid phase.Assay Format #2

[0220] In some embodiments, after washing the Biomarker capture particles or solid phase with Wash Buffer (0.05-0.10% Tween-20, 150-200 mM NaCl, 10-20 mM TRIS, and pH 7.0- 8.0) and / or with specific binding buffer (mildly alkaline pH such as pH 7.5 to 8.5, 100-200 mM sodium chloride, and 0.05 to 0.10% Tween-20) to remove the sample matrix as well as any normal Tau from the PAT-Tau complex captured by the Biomarker capture particles, the PAT can subsequently be eluted from the Biomarker capture particles or solid phase using an Elution Buffer such as 0.1M Glycine, 0.05-0.10% Tween-20, pH 2.5, or 0.05M Acetic Acid, 0.05-0.10% Tween-20, pH 3.1, to disrupt the affinity binding interaction of the anti-PAT or anti-PAT-Tau antibody or capture moiety. In some embodiments, after elution of PAT, the magnetic beads areremoved or isolated from the eluate via magnetic separation, filtration, or centrifugation, and the eluate is neutralized with a Neutralization Buffer, e.g., 0.3M TRIS, 0.05-0.10% Tween-20, pH 10.0-10.5. In some embodiments, the PAT can be eluted or cleaved from the Biomarker capture particles using a smaller volume of Elution Buffer, e.g., 100 to 200 pL, whereby the total PAT biomarkers have been purified and enriched or concentrated in 100-200 pL Elution Buffer. In some embodiments, immediately after total PAT elution from the Capture Beads with 100-200 pL Elution Buffer, the eluate is neutralized with a Neutralization Buffer such as 13-26 pL 300 mL TRIS pH 10.5. In some embodiments, for each wash of the Biomarker capture particles, the capture beads can be mixed, homogenously resuspended, and incubated in the Wash Buffer for 1-5 min at 37-40 degrees Celsius to improve wash efficiency but also recapture any PAT that can have come off the bead surface during the addition of the Wash Buffer to maximize PAT recovery. In some embodiments, if VeraBIND is used to capture, wash, elute, and neutralize PAT or PAT-Tau from the biological fluid or CSF, plasma, serum, DBS, pDBS plasma, saliva, saline oral rinse, or urine the VeraBIND purified PAT or PAT-Tau biomarker will be a VeraBIND matrix-free sample for subsequent immunoassay detection either using a conventional solid phase heterogeneous (wash) sandwich or competitive assay formats, or subsequent immunoassay detection using the Lumit® homogeneous (no wash) assay format whereby a SmTrip labeled antibody pair against PAT or PAT-Tau, or SmTrip labeled antibody and Tau peptide, such that the assay will generate a signal response only when the Lumit™ SmTrip9 and SmTriplO labeled pair binds PAT or PAT-Tau.Assay Format #3

[0221] In some embodiments, the total PAT or PAT-Tau captured by the antibodies or capture moieties further comprise complexed or pre-bound PAT to Normal Tau. In some embodiments, the Wash Buffer can further be optimized to wash, cleave, disrupt, elute, or dissociate any Normal Tau from PAT from the Capture Beads such that the only protein or peptide remaining on the Capture bead surface after washing is total PAT without any bound Normal Tau. In some embodiments, if normal Tau can be eluted from the PAT-Tau complex captured by the anti-PAT or anti-PAT-Tau Biomarker capture particles, e.g., by first washing the capture beads with the Specific Binding Buffer to promote PAT binding to normal Tau during these washes to remove the sample matrix such as a buffer with a mildly alkaline pH such as pH 7.5 to 8.5, 100-200 mM sodium chloride, and 0.05 to 0.10% Tween-20, then the normal Tau can be specifically eluted from the PAT-Tau complex using a wash buffer that will only disrupt PAT binding to normal Tau but not anti-PAT binding to PAT, e.g., a wash buffer with an acidic pH less than pH 7.0, a high salt, (e.g., 1000-2000 mM sodium chloride), Triton X-100 greater than0.2% (v / v), 0.05 to 0.010% Tween-20, and a reducing agent, e.g., DTT or TCEP to cleave disulfide bonds instrumental in the initiation of Tau aggregation. In some embodiments, if VeraBIND captures the PAT-Tau complex from samples collected from patients with AD pathology, then normal Tau elutes for subsequent detection. In some embodiments, if VeraBIND captures the PAT-Tau complex from samples collected from patients with AD pathology, then normal Tau elutes for subsequent detection, wherein a positive screening or test result for AD Tau pathology or Tau tangles. In some embodiments, the VeraBIND purified normal Tau biomarker will be a VeraBIND matrix-free sample for subsequent immunoassay detection either using a conventional solid phase heterogeneous (wash) sandwich or competitive assay formats, or subsequent immunoassay detection using the Lumit® homogeneous (no wash) assay format whereby a SmTrip labeled antibody pair against normal Tau, or SmTrip labeled antibody and phosphorylated Tau peptide or PAT, such that the assay will generate a signal response only when the Lumit™ SmTrip9 and SmTriplO labeled pair binds normal Tau.Assay Format #4

[0222] In some embodiments, PAT conformation or tertiary structures in patients with AD Pathology differ than PAT conformation or tertiary structures in healthy controls. In some embodiments, the VeraBIND Sample “Matrix Free Buffer” will comprise purified and enriched PAT. In some embodiments, if normal Tau, or a recombinant peptide of normal Tau that comprises the amino acid sequence whereby PAT binds to Normal Tau, is added to the sample to demonstrate normal Tau binding to PAT, the Assay Buffer comprises cofactors or chemicals, e.g., divalent metal ions like zinc, magnesium, or copper, to promote the correct orientation or tertiary structure of the purified and enriched PAT such that is will only bind to Normal Tau or a recombinant peptide, or plurality of Tau peptides, then the PAT is in the correct or disease specific conformation only in the presence of AD pathology.

[0223] In some embodiments, the Assay Buffer comprises the Specific Binding Buffer, or a buffer with a mildly alkaline pH such as pH 7.5 to 8.5, 100-200 mM sodium chloride, and 0.05 to 0.10% Tween-20 that was used to capture PAT or PAT-Tau with Biomarker capture particles, to promote the binding of the normal Tau or recombinant Tau peptide to the VeraBIND purified PAT. In some embodiments, the binding interaction can be detected and measured using Lumit™ detection technology if SmTrip labeled antibody and Tau peptide is added the purified PAT in Specific Binding Buffer, with or without cofactors or chemicals, e.g., divalent metal ions like zinc, magnesium, or copper, to promote the correct orientation or tertiary structure of the purified and enriched PAT.

[0224] In some embodiments, if the SmTrip 9-labeled normal Tau binds to the PAT, and ananti-PAT antibody labeled with SmTriplO also binds to the same PAT, then Lumit™ the detection reagents, e.g., LgTrip, Furimazine substrate, generate a signal response. In some embodiments, the anti-PAT antibody can be a polyclonal or monoclonal antibody specific to a phosphorylated amino acid in PAT such as pTau217, or it can be a conformation-specific monoclonal antibody that only detects PAT. In some embodiments, the purified PAT comprises the sample, and the Lumit™ technology is used for a no-wash proximity based homogeneous assay whereby the recombinant normal Tau peptide(s) is labeled with Lumit™ SmTrip9 or 10, and the anti -normal Tau or anti-Tau tag or fusion protein antibody is labeled with the complementary SmTrip9 or 10. In some embodiments, the assay signal will be directly proportional to the amount of normal Tau / PAT complex that forms.Assay Format #5

[0225] In some embodiments, the method of assay formatting comprises, after capturing the total PAT by Biomarker capture particles and washing the beads with Wash Buffer, adding labeled Normal Tau peptide, e.g., HRP, ALP, ruthenium, chemiluminescent substrate such as acridinium, luminol, or ABIE, Fluorophore, Lumit, etc., or peptides to bind to the PAT on the solid phase, incubating, washing, and reading the assay signal. In some embodiments, the amount of signal detected will be directly proportional to the amount of normal Tau bound to PAT on the beads.

[0226] In some embodiments, the method of assay formatting comprises adding tagged, or recombinant tagged normal Tau to bind to any PAT on the beads, incubating, washing, adding anti -tag, recombinant tag, or fusion protein specific monoclonal antibody conjugate, incubating, washing, and reading the assay signal. In some embodiments, the method of assay formatting comprises adding tagged, or recombinant tagged normal Tau to bind to any PAT on the beads, incubating, washing, adding anti-tag, recombinant tag, or fusion protein specific monoclonal antibody conjugate, incubating, washing, eluting, neutralizing, and reading the assay signal. In some embodiments, the signal detected is directly proportional to the amount of anti-tag or antifusion protein antibody conjugate that binds to normal Tau on the beads, and directly proportional to the amount of normal Tau that has bound to PAT on the beads. In some embodiments, the Lumit™ detection system can be used if the normal Tau is labeled with SmTrip 9 or 10, either covalently via ester chemistry or recombinantly via insertion in the plasmid, and the anti -tag antibody is labelled with the complementary SmTrip9 or 10, either covalently via ester chemistry or recombinantly via insertion in the clone.

[0227] In some embodiments, the VeraBIND™ (Biomarker Isolation and N-enrichment for Detection) sample transformation and biomarker purification technology is used to develop asensitive and specific AD screening test to (a) detect hyperphosphorylated Tau binding to normal Tau, or detect the complex or aggregate of hyperphosphorylated Tau bound to normal Tau, in blood-based or saliva-based samples collected from pre-symptomatic and symptomatic patients as a biomarker of Alzheimer’s pathology and related tauopathies, and (b) to avoid the effect of sample matrix interference on sensitivity and specificity of the assay by removing potential substances pre-analytically that can cause false positive or negative results (FIG. 1 and FIG. 2)

[0228] FIG. 1 shows the adverse effects of interferences and sample matrix on patient samples, a challenge faced by assay manufacturers and CLIA labs when developing and commercializing diagnostics tests. Heterophilic interference and sample matrix effects can cause false negatives and / or positives, skewing the testing results.

[0229] FIG. 2 shows an example process for processing a patient sample. The patient sample can be any patient sample type. In some embodiments, the patient sample can be blood. In some embodiments, the patient sample can be plasma. In some embodiments, the patient sample can be cerebrospinal fluid. In some embodiments, the patient sample can be dry blood spots. In some embodiments, the patient sample can be saliva. In some embodiments, the patient sample can be saline oral rinse (SOR). In some embodiments, the patient sample can be stool. In some embodiments, the patient sample can be urine. First, the patient sample can be inserted into a collection tube. Then, the interference capture particles can be added into the collection tube to remove interferences within and improve specificity of the patient sample. In some embodiments, the interference capture particles can be magnetic beads. Next, biomarker capture particles isolate and concentrate biomarkers to improve sensitivity. In some embodiments, the biomarker capture particles can be beads. In some embodiments, the biomarker capture particles can be magnetic beads. Then, the concentrated biomarkers are released or eluted from the biomarker capture particles, forming a new sample containing only the biomarkers of interest. The new sample can be tested for the biomarkers (e.g., presence / absence, concentration, amount, etc.) using any detection method. In some embodiments, the detection method can be an immunoassay. In some embodiments, the detection method can be a molecular detection method. In some embodiments, the detection method can be a polymerase-chain-reaction. In some embodiments, the detection method can be any type of spectrometry. In some embodiments, the detection method can be mass spectrometry. In some embodiments, the detection method can be any existing assay. In some embodiments, the detection method can be Biolayer interferometry (BLI) and surface plasmon resonance (SPR) or label-free optical techniques for measuring biomolecular interactions.

[0230] In some embodiments, the interference capture particles are designed to selectively target and pre-analytically remove various potential sources of sample-specific interference that can impact the accuracy of immunoassay results. In some embodiments, the Interference capture particles are highly sensitive and specific diagnostic assays capable of detecting low abundance biomarkers for a wide range of pathogenic and non-pathogenic diseases.

[0231] In some embodiments, the VeraBIND sample cleaning step uses interference capture particles and involves the removal of interference substances, e.g., Human anti-mouse antibody interference (HAMA), Rheumatoid factor interference (RF), Human anti-animal antibody interference (anti-mouse, goat, rabbit, sheep, and bovine IgG interference), anti-histidine interference, Avidin / biotin interference (anti-streptavidin, anti-biotin, and free biotin interference), Anti-PEG interference, Anti-PVP interference, autoantibodies against Tau and pTau, and Macro-complexes of autoantibodies bound to Tau and pTau, anti-detection moiety interference (such as human anti-ALP, anti -ruthenium, anti-fluorescein, anti-ABEI interference), free biotin interference, as well as other non-specific binding (NSB) interference or solid phase specific interference, that could potentially lead to false positive or false negative test results. In some embodiments, magnetic nanobeads conjugated with proprietary cocktails of antibodies and other proteins are used for the removal of interference substances. In some embodiments, after magnetic nanobeads conjugated with proprietary cocktails of antibodies and other proteins are added to the sample, mixed to form a homogenous suspension, and incubated with the sample, they are subsequently removed or isolated from the sample by magnetic separation, filtration, or centrifugation. In some embodiments, removal or isolation from the sample by magnetic separation, filtration, or centrifugation. Is crucial for ensuring the accuracy and reliability of the subsequent biomarker capture, purification, enrichment, and detection (FIG. 3).

[0232] In some embodiments, after the interference capture particle cleaning step, biomarker capture particles, superparamagnetic biomarker-specific antibody-coated nanobeads, or superparamagnetic biomarker-specific peptide coated nanobeads can be used to capture the target biomarkers. In some embodiments, after subsequent sample incubation and magnetic separation, the targeted capture enhances the concentration of biomarkers making them more detectable in subsequent analyses. In some embodiments, two or more different Biomarker capture particles, each coated with a different capture moiety, are pooled for multiplex biomarker capture, purification, and enrichment from the same sample during the same reaction. In some embodiments, the captured biomarkers can be eluted from the Biomarker capture particles using a matrix-free buffer. In some embodiments, the elution process increases the concentration of the biomarkers, enabling more sensitive detection in follow-up analyses. Insome embodiments, purified and enriched biomarkers from plasma, dried blood spots, capillary plasma, saliva, and SOR can subsequently be detected with high sensitivity and high specificity.

[0233] In some embodiments, the method of assay formatting comprises maintaining consistency and high performance of the antibodies used. In some embodiments, the method of assay formatting comprises SEC-HPLC analysis to select monomeric antibodies for conjugations. In some embodiments, the monomeric antibodies are then biotinylated, purified by SEC-HPLC, and conjugated to Streptavidin Beads using a coating process and equipment to prepare both Interference capture particles and biomarker capture particles. In some embodiments, this approach ensures the reliability and accuracy of the AD screening test throughout its development and commercialization stages (FIG. 4).

[0234] In some embodiments, the ability to consistently detect traces of biomarkers, or low abundance biomarkers in the femtogram per milliliter (fg / mL) range, plays a pivotal role in the successful development of a screening test, particularly when targeting specimens containing ultra-low levels of the biomarkers of interest. In some embodiments, the Promega Inc., Lumit™ proximity-based homogeneous bioluminescence detection technology is based on a luminescent structural complementation system consisting of two small peptide tags and a reagent-based polypeptide (LgT). In some embodiments, the Lumit™ Immunoassay, antibodies are chemically labeled with the peptide tags SmTrip9 and SmTriplO. In some embodiments, in the presence of target analyte, the labeled antibodies bind the protein, bringing the peptide subunits into close proximity allowing the binding of LgT present in solution to reassemble into a functional luminescent enzyme that generates a luminescent signal in the presence of furimazine substrate. In some embodiments, the assay does not require immobilization of detection antibodies to plate, beads, or other surfaces. In some embodiments, add Lumit™ reagents to the sample, wait for 90 minutes or less, add detection reagent, and read.

[0235] In some embodiments, the method of assay formatting comprises using bioluminescence-based detection to improve test specificity by minimizing false positive signals arising from potential interference, wherein the assay signal is highly specific to the fixed proximity-based binding of the bioluminescence-based detection SmTrip9 and SmTriplO labeled antibody pair to their antigen. In some embodiments, when the bioluminescence-based detection technology is combined with the sample transformation and biomarker purification technology disclosed herein, by which pre-analytically targets and removes HAMA and RF interference with the Interference capture particles and a subsequent washes away the sample matrix after the Biomarker capture particles capture the biomarkers, the test specificity is further enhanced to mitigate the possibility of any false positive signal or false positive results. In someembodiments, bioluminescence-based detection improves test sensitivity and minimizes false negative signal and false negative results due to the high sensitivity of the bioluminescence signal generated by the binding of LgT present in solution to reassemble into a functional luminescent enzyme that generates a luminescent signal in the presence of furimazine substrate. In some embodiments, when bioluminescence-based detection is combined with the sample transformation and biomarker purification technology disclosed herein, by which the Biomarker capture particles capture, purify, and enrich the biomarkers into a “matrix free” sample which further improves assay signal-to-noise and analytical sensitivity, the test sensitivity is further enhanced to mitigate the possibility of any false negative signal or false negative results (FIG. 5).

[0236] In some embodiments, the sample transformation and biomarker purification technology are designed to capture, purify, and concentrate low levels of AD biomarkers, or to enrich low abundance AD biomarkers, for their subsequent detection which are essential for identifying the early stages of the disease. In some embodiments, the use of Interference capture particles during the sample purification process improves the specificity, sensitivity, and signal- to-noise of the subsequent assay or test by removing interferences that can contribute to high assay background signal (false positive signal) and decreased analyte signal (false negative signal) by binding to the capture beads and causing steric hindrance, blocking access to capture bead’s surface antibodies. In some embodiments, subsequent Biomarker capture particles capture of the biomarkers and removal of the sample matrix via magnetic bead washing further enhances the specificity and sensitivity of the detection method. In some embodiments, the methods disclosed herein remove potential false positive or negative results caused by samplespecific interference or the sample matrix interference. In some embodiments, eliminating interference pre-analytically optimizes and enhances the assay’s specificity and accuracy in detecting and measuring accurate biomarker levels, and the subsequent femtogram-level (fg / mL) detection sensitivity of the Lumit™ immunoassay highlights the capability of the methods disclosed herein to identify even the smallest quantities of AD biomarkers with high sensitivity and specificity.

[0237] In some embodiments, the use of Interference capture particles, biomarker-specific capture beads, and capture bead washing provides the first degree of specificity and sensitivity, and the biomarker-specific bioluminescence-based detection immunoassay provides the second degree of specificity and sensitivity, for combined ultra-sensitive and ultra-specific detection and measurement of biomarkers from challenging sample types, e.g., plasma, DBS, saliva, and saline oral rinse.

[0238] In some embodiments, the use of the VeraBIND sample transformation and biomarker purification enhances biomarker detection and offers the potential to identify early signs of AD, e.g., the detection of hyperphosphorylated Tau binding to normal Tau, or detection of the hyperphosphorylated Tau-normal Tau complex. In some embodiments, the ability to detect multiple biomarkers simultaneously is a powerful feature of the VeraBIND bead-based technology as AD is a complex disease with multiple biomarkers. In some embodiments, the VeraBIND bead-based technology being able to capture and measure multiple biomarkers at once enhances diagnostic efficiency and provides a more comprehensive view of disease progression. In some embodiments, the ability of the methods disclosed herein to work with easy to collect and accessible specimens align with the goal of enabling at-home sample collection, which would greatly facilitate routine screening. In some embodiments, the compatibility of the methods disclosed herein with different sample types, including blood, plasma, serum, DBS, capillary collection, saliva, saline oral rinse, and urine, allows versatility for more widespread usage and can cater to individual preferences and situations.

[0239] In some embodiment, detection is label free such as Biolayer interferometry (BLI) and surface plasmon resonance (SPR) which are both label-free optical techniques for measuring biomolecular interactions. Detection is based on the binding interaction of normal tau to HPT on the sensor surface. For example, anti-pTau antibodies can be coated on the sensor and washed, the patient sample added and washed for the capture of HPT by the anti-pTau antibodies on the sensor, the normal tau added and washed for binding with any HPT on the sensor, and this biomolecular interaction detected and measured.

[0240] In some embodiments, a detection label can comprise a fluorophore, a stable isotope, a mass tag, horse radish peroxidase (HRP), alkaline phosphatase (ALP), Luciferase, chemiluminescent substrate such as isoluminol, luminol, acridinium ester, or ABEI, an electrochemiluminescence substrate such as ruthenium, a peptide such as Lumit® SmTrip9 or SmTriplO, oligonucleotide, a recombinant tag such as a polyhistidine tag (e.g., His-tag, an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag), a V5 tag (a small epitope tag GKPIPNPLLGLDST; SEQ ID NO: 23, which can be placed by molecular cloning at the N- or C-terminus of a protein of choice), a HA tag (YPYDVPDYA; SEQ ID NO: 24), a FLAG tag (DYKDDDDK; SEQ ID NO: 25), a c-Myc tag (EQKLISEEDL; SEQ ID NO: 26), a GSK tag (26 kDa sequence of 211 amino acids), an immuno-PCR oligonucleotide labeled antibody or peptide, Tandem mass tagged antibodies or peptides (there are currently six varieties of TMT available: TMTzero, a non-isotopicallysubstituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution; TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions; 10- plex - a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da, TMTpro a 16 pl ex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero), stable isotope labelled antibodies or peptides (e.g., peptides labeled with light isotopes (XH,12C,14N, and / or16O) are combined with peptides labeled with heavy isotopes (2H,13C,15N, and / or18O) prior to mass spectrometry (MS) and the relative peak intensity of the two, or a combination thereof.

[0241] In some embodiments, an anti-PAT capture moiety or anti-PAT-Tau complex capture moiety can comprise an anti-hyperphosphorylated antibody against a specific phosphorylation site (e.g., Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404 or Ser422), an antibody against a conformation of a PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer’s disease pathology, an antibody against a PAT-Tau complex, an antibody against a conformation of a PAT protein or a portion or fragment thereof when a PAT is in a PAT-Tau complex, non-hyperphosphorylated Tau such as recombinant human Tau-441, or a fragment or portion of tau441 peptide, e.g., comprising the MTBR R1-R4, or anti-PAT polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2 antibody fragment, molecular imprinted polymer (MIP), aptamer, alpaca nanobody or llama derived nanobody, tau specific PAT capture antibody such as clones against total Tau such as clone RD-073 (amino acids 1- 20), clone HT7 (aa 159-163), and clone BT2 (aa 194-198), or clones against the microtubule binding region (MTBR) such as MTBR-tau243 based monoclonal antibody clones HJ32.11 (aa 225-242 with proximity to aa 243) or HJ34.8 (aa 226-264 with proximity to aa 260), or monoclonal antibody clone 77G7 against MTBR (the epitope mapping and sequence have been provided; residues 256-273 (VKSKIGSTENLKHQPGGG, SEQ ID NO: 29) or more specifically aa 268-271 (HQPG), aa residues 287-304 (VQSKCGSKDNIKHVPPGG, SEQ ID NO: 30) or more specifically aa 299-302 (HVPG), aa residues 318-335 (VTSKCGSLGNIHHKPGGG, SEQ ID NO: 31) or more specifically aa 330-333 (HKPG), aa residues 350-364 (VQSKIGSLDNITHVPGGG, SEQ ID NO: 32) or more specifically aa 362-365 (HVPG)).

[0242] In some embodiments, two or more different capture moieties, or a plurality of capture moieties, can be co-coated on a beads or solid phase, or two or more different capture moiety coated beads can be pooled, to maximize recovery of total PAT or PAT-Tau complex captured from a biological specimen such as antibody clone RD-085 from ADX Neurosciencesspecific for pTau217 and antibody clone ADx253 from ADX Neurosciences and antibody clone ATI 80 from Fujirebio which are both monoclonal antibodies specific for pTau231, but specific to different immunogens. There is debate on the epitope of ATI 80: the core epitope is around pT231 but the need of pT235 is uncertain. The reason is that ATI 80 is generated with PHF tau while ADx253 is generated with synthetic peptide-based antigen. In any case independent of the epitope, in bead-based assays the ADx253 antibody has performed best regarding signal to noise values.

[0243] In some embodiments, a binding of PAT or PAT-Tau complex to nonhyperphosphorylated Tau such as Tau-441 can be enhanced using a binding buffer with a mildly alkaline pH such as pH 7.5 to 8.5, 100-200 mM sodium chloride, and 0.05 to 0.10% Tween-20, and by incubating at an elevated temperature such as 37-40 degree Celsius.

[0244] In some embodiments, a PAT detection moiety or PAT-Tau detection moiety can bind a different PAT or PAT-Tau complex site, epitope, or conformation as a capture moiety, and a detection moiety can comprise a labeled anti-hyperphosphorylated antibody against a specific phosphorylation site (e.g., Thrl81, Serl99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404 or Ser422), a labelled antibody against a conformation of a PAT protein or a portion or fragment thereof specific to tau pathology and Alzheimer’s disease pathology, a labelled antibody against a PAT-Tau complex, a labelled antibody against a conformation of a PAT protein or a portion or fragment thereof when a PAT is in a PAT-Tau complex, a labelled non-hy perphosphorylated Tau such as recombinant human Tau-441, or a fragment or portion of tau441 peptide, e.g., comprising the MTBR R1-R4, or a labelled anti- PAT or PAT-Tau complex polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2 antibody fragment, molecular imprinted polymer (MIP), aptamer, alpaca nanobody or llama derived nanobody.

[0245] In some embodiments, two or more different detection moieties, or a plurality of detection moieties, can be pooled to maximize detection of total PAT or PAT-Tau complex captured from a biological specimen.

[0246] In some embodiments, a binding of a non-hyperphosphorylated Tau detection moiety to PAT, such as Tau-441 to PAT, or a fragment or portion of tau441 peptide comprising the MTBR R1-R4, can be enhanced using a binding buffer with a mildly alkaline pH such as pH 7.5 to 8.5, 100-200 mM sodium chloride, and 0.05 to 0.10% Tween-20, and by incubating at an elevated temperature such as 37-40 degree Celsius.

[0247] In some embodiments, a binding of Tau-441 to PAT, or a fragment or portion of tau441 peptide comprising the MTBR R1-R4, can be detected by using Tau-441 or a fragmentor portion of tau441 peptide comprising the MTBR R1-R4 labelled with a fluorophore, a stable isotope, a mass tag, horse radish peroxidase (HRP), alkaline phosphatase (ALP), Luciferase, chemiluminescent substrate such as isoluminol, luminol, acridinium ester, or, ABEI, an electrochemiluminescence substrate such as ruthenium, a peptide such as Lumit® SmTrip9 or SmTriplO, oligonucleotide, or a combination thereof.

[0248] In some embodiments, a binding of Tau-441 to PAT, or a fragment or portion of tau441 peptide comprising the MTBR R1-R4, can be detected using a labelled anti-Tau-441 or a fragment or portion of tau441 peptide comprising the MTBR R1-R4 polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2 antibody fragment, molecular imprinted polymer (MIP), aptamer, alpaca nanobody or llama derived nanobody specific to an epitope or binding site of Tau-441, or specific to a Tau-441 recombinant tag or fusion protein, or specific to an epitope or binding site to a fragment or portion of tau441 peptide comprising the MTBR R1-R4, or specific to a fragment or portion of tau441 peptide comprising the MTBR R1-R4 recombinant tag or fusion protein.

[0249] In some embodiments, a binding of Tau-441, or specific to an epitope or binding site to a fragment or portion of tau441 peptide comprising the MTBR R1-R4, to PAT can be detected using Tau-441 or specific to an epitope or binding site to a fragment or portion of tau441 peptide comprising the MTBR R1-R4 labelled with a Lumit® SmTrip peptide tag and an anti-Tau-441 detection moiety labelled with a Lumit® SmTrip peptide tag, or a fragment or portion of tau441 peptide comprising the MTBR R1-R4 labelled with a Lumit® SmTrip peptide tag, whereby in a presence of target PAT analyte, a labeled antibodies bind a protein, bringing a peptide subunits into close-proximity allowing a binding of LgTrip present in solution to reassemble into a functional luminescent enzyme that generates a luminescent signal in a presence of furimazine substrate.

[0250] In some embodiments, a non-hyperphosphorylated Tau detection moiety should bind a different site, epitope, or conformation of Tau then a PAT or PAT-Tau complex capture moiety and can be a labelled polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2 antibody fragment, molecular imprinted polymer (MIP), aptamer, alpaca nanobody or llama derived nanobody.

[0251] In some embodiments, after capture of a PAT-Tau complex by a capture moiety coated solid phase, a non-hyperphosphorylated Tau can be eluted from a PAT using a buffer with an acidic pH less than pH 7.0, a high salt such as 1000-2000 mM sodium chloride, Triton X-100 greater than 0.2% (v / v), 0.05 to 0.010% Tween-20, and a reducing agent such as DTT orTCEP to cleave disulfide bonds instrumental in an initiation of Tau aggregation.

[0252] Eluted non-phosphorylated Tau can be detected with a labelled anti-Tau polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab, Fab’2, chimeric antibody, chimeric Fab’2 antibody fragment, molecular imprinted polymer (MIP), aptamer, alpaca nanobody or llama derived nanobody.

[0253] In some embodiments, the PAT can be eluted from the capture moiety solid phase and neutralized for the subsequent detection of PAT.

[0254] In some embodiments, the PAT can be purified prior to detection. In some embodiments, the PAT can be eluted from the capture moiety solid phase after first washing the solid phase to elute or remove any non-hyperphosphorylated Tau from PAT-Tau complex. The eluted purified PAT can be further neutralized for the subsequent detection of the purified PAT.

[0255] In some embodiments, the PAT or the purified PAT can be detected using a binding pair comprising a capture moiety coated solid phase against PAT and a labeled detection moiety against PAT as disclosed herein.

[0256] In some embodiments, the PAT or the purified PAT can be detected using the Lumit proximity based homogeneous bioluminescence detection technology with the following reagents added to the PAT sample or purified PAT sample: Tau-441 (or a fragment or portion of tau441 peptide comprising the MTBR R1-R4) labelled with a Lumit® SmTrip peptide tag and an anti- PAT-Tau-441 complex antibody, or an anti- PAT-Tau-441 conformation-specific antibody, labelled with a Lumit® SmTrip peptide tag. In the presence of PAT in the sample, Tau-441 -SmTrip can bind to PAT to form PAT-Tau-441 complex, and the antibody can subsequently bind to the PAT-Tau-441 complex but not bind to any free Tau-441 -SmTrip or any free PAT. This highly specific binding complex of [anti-PAT-Tau-441-SmTrip Antibody]- (PAT-Tau-441-SmTrip) can bring the SmTrip9 and SmTriplO peptide subunits into closeproximity, allowing the binding of LgTrip present in sample to reassemble into a functional luminescent enzyme that generates a luminescent signal in the presence of furimazine substrate.

[0257] In some embodiments, after capturing PAT using the biomarker capture particles and incubating it with normal Tau, the PAT and normal Tau on the complex can be cross-linked to maintain the interaction between PAT and Tau throughout subsequent washing steps. In some embodiments, the cross-linking comprises adding a cross link agent. In some embodiments, the cross-linking agent can comprise glutaraldehyde or formaldehyde.

[0258] In some embodiments, the method can further comprise cross linking the PAT-Tau complex.Plate Blocking Composition

[0259] In some embodiments, the present disclosure provides a plate blocking composition or buffer. In some embodiments, the plate blocking composition can comprise a blocking reagent.

[0260] Non-specific binding (NSB) of proteins or other biomolecules to unoccupied spaces on a surface of a plate (e.g., a plastic plate with wells) that is used in an assay can be detrimental to the specificity and sensitivity of the assay results. Tau is a sticky protein and can bind hydrophobically or non-specifically to non-polypropylene (PP) plastics such as low-density polyethylene (LDPE), high density polyethylene (HDPE), and polystyrene (PS) plastics (The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid [3 and tau. Alzheimers Dement. 2021 Sep;17(9): 1575-1582. doi: 10.1002 / alz.l2316. Epub 2021 Mar 31, which is incorporated herein by reference). Nonspecific binding of proteins or other biomolecules to the surface of the plate can be minimized by saturating these unoccupied binding sites with a blocking reagent. In some embodiments, a blocking reagent can comprise a casein sodium salt. In some embodiments, a block reagent can comprise 2% (w / v) casein sodium salt (e.g., from bovine milk) in NTBB (10 mM Tris-HCl, 200 mM NaCl, 0.10% Tween® 20, 0.05% sodium azide, pH 7.6) to mitigate non-specific binding (NSB), loss and under recovery of sample-specific HPT to the surface of the wells of the plate, and to block NSB of the assay Normal Tau reagent (e.g., Tau-441 with V5 tag) to the surface of the wells. Although casein, a non-fat dry milk (NFDM) component, can be used as a stable blocking reagent (primarily for DNA blots), NFDM tends to be more dispersible in aqueous buffers than pure casein. Casein has very poor solubility in aqueous buffers and a method is needed to improve the dispersion of casein in aqueous buffers. In some embodiments, casein can be mixed and solubilized into a homogeneous suspension in aqueous buffer with up to 3% (w / v) concentration using sonication with mixing. In some embodiments, sonication time can be closely monitored as over-sonication can decrease blocking efficiency of the casein by over fragmenting or degrading the casein. Other form of fragmented casein such as Casein hydrolysate are not effective blocker compared to 2% (w / v) casein sodium salt.

[0261] The 2% (w / v) casein sodium salt solution can be prepared by gradually adding 60 g of casein sodium salt to a 5000 mL glass flask containing 2000 mL of NTBB, with constant mixing using an overhead propeller at 300 rpm. The resulting suspension can be sonicated using a sonication bath for 20 minutes, followed by an increase in propeller speed to 400 rpm for an additional 20 minutes of sonication. The speed can be further increased to 450 rpm, and the suspension can be sonicated for another 30 minutes, totaling 70 minutes of mixing and sonication. The resulting clear solution can be diluted to 2% casein by adding 1000 mL ofNTBB, followed by 3 hours of additional mixing at 300 rpm without sonication. After this, the solution can be transferred to a glass bottle and stored at 4°C.

[0262] In some embodiments, a plate blocking protocol can comprise adding plate blocking buffer to a plate (e.g., Coming® 96-well White Round Bottom Polystyrene NBS Microplate) and incubating the plate for at least 60 min or until the plate is used. In some embodiments, the plate can be aspirated with the plate blocking buffer. In some embodiments, the aspirating can be performed with a pipette (e.g., an automatic or electric pipette). In some embodiments, the plate can be washed with a wash buffer and incubated for at least 1 min at 1000 rpm. After the washing and discarding of supernatant, the plate can be dried, e.g., by placed inverted on a dry paper towel for drain left over buffer.

[0263] In some embodiments, a blocking reagent can comprise a non-ionic detergent such as Tween-20 for hard plastic assays (e.g., 96-well microplates or strips). In some embodiments, Tween-20 can be present in the plate blocking buffer at a concentration ranging from 0.01% to 0.1% (v / v). In some embodiments, the non-ionic detergent can block non-specific binding to the surface. In some embodiments, the non-ionic detergent cannot affect specific binding to the surface. In some embodiments, hydrophobic surfaces can have medium binding activity. In some embodiments, hydrophobic surfaces can be effectively blocked with either non-ionic detergents or protein blockers. In some embodiments, surfaces that are comprised of hydrophobic and ionic binding sites can have high binding activity. High binding surfaces are slightly more difficult to block than medium binding surfaces. The combined use of a non-ionic detergent (Tween-20) and a protein blocker (1% BSA, 0.2% NFDM, 10% normal sera, 1-3% Casein, etc.) can effectively minimize non-specific binding. The choice of protein blocker is more dependent on the assay’s reactive biomolecules than on the surface itself. In some embodiments, 0.10% Tween-20 (v / v) when used in combination with 2% Casein (w / v) results in better overall plate blocking and the lowest HPT and Normal Tau NSB.Pre-conditioning Composition

[0264] In some embodiments, the present disclosure provides a composition for preconditioning a sample. In some embodiments, the pre-conditioning can be performed before contacting the sample with a plurality of interference capture particles (e.g., Clean Beads). In some embodiments, the composition can comprise a first reagent. In some embodiments, the first reagent can inactivate or reduce an activity of one or more endogenous enzymes. In some embodiments, the one or more endogenous enzymes can deactivate a biomarker. In some embodiments, the deactivation of the biomarker can result in no detection of the biomarker, thereby resulting a false negative result. In some embodiments, inactivating or reducing anactivity of the one or more endogenous enzymes can improve the sensitivity of the assay or detection of the biomarker. In some embodiments, inactivating or reducing an activity of the one or more endogenous enzymes can reduce the likelihood of a false negative result. In some embodiments, the one or more endogenous enzymes can deactivate or degrade a physiologically active protein (e.g., a disease protein). In some embodiments, the one or more endogenous enzymes can deactivate or degrade a hyperphosphorylated protein (e.g., PAT). In some embodiments, the one or more endogenous enzymes can de-phosphorylate a hyperphosphorylated protein (e.g., PAT). In some embodiments, dephosphorylation of HPT can abolish or reduce the ability of HPT to aggregate with normal tau and / or form tangles. In some embodiments, the first reagent can prevent or reduce the deactivation or degradation of the physiologically active protein. In some embodiments, the first reagent can prevent or reduce the dephosphorylation of HPT. In some embodiments, the first reagent can comprise a chelating agent. In some embodiments, the first reagent can comprise ethylenediaminetetraacetic acid (EDTA). In some embodiments, the first reagent can comprise a salt of EDTA (e.g., sodium or calcium salt of EDTA). In some embodiments, the first reagent (e.g., EDTA) can be present in the composition from about 1 mM to about 2 mM, from about 1 mM to about 3 mM, from about 1 mM to about 4 mM, from about 1 mM to about 5 mM, from about 1 mM to about 7.5 mM, from about 1 mM to about 10 mM, from about 1 mM to about 12.5 mM, from about 1 mM to about 15 mM, from about 1 mM to about 20 mM, from about 1 mM to about 25 mM, from about 1 mM to about 30 mM, from about 1 mM to about 40 mM, from about 1 mM to about 50 mM, from about 2 mM to about 3 mM, from about 2 mM to about 4 mM, from about 2 mM to about 5 mM, from about 2 mM to about 7.5 mM, from about 2 mM to about 10 mM, from about 2 mM to about 12.5 mM, from about 2 mM to about 15 mM, from about 2 mM to about 20 mM, from about 2 mM to about 25 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 50 mM, from about 3 mM to about 4 mM, from about 3 mM to about 5 mM, from about 3 mM to about 7.5 mM, from about 3 mM to about 10 mM, from about3 mM to about 12.5 mM, from about 3 mM to about 15 mM, from about 3 mM to about 20 mM, from about 3 mM to about 25 mM, from about 3 mM to about 30 mM, from about 3 mM to about 40 mM, from about 3 mM to about 50 mM, from about 4 mM to about 5 mM, from about4 mM to about 7.5 mM, from about 4 mM to about 10 mM, from about 4 mM to about 12.5 mM, from about 4 mM to about 15 mM, from about 4 mM to about 20 mM, from about 4 mM to about 25 mM, from about 4 mM to about 30 mM, from about 4 mM to about 40 mM, from about 4 mM to about 50 mM, from about 5 mM to about 7.5 mM, from about 5 mM to about 10 mM, from about 5 mM to about 12.5 mM, from about 5 mM to about 15 mM, from about 5 mM toabout 20 mM, from about 5 mM to about 25 mM, from about 5 mM to about 30 mM, from about 5 mM to about 40 mM, from about 5 mM to about 50 mM, from about 7.5 mM to about 10 mM, from about 7.5 mM to about 12.5 mM, from about 7.5 mM to about 15 mM, from about 7.5 mM to about 20 mM, from about 7.5 mM to about 25 mM, from about 7.5 mM to about 30 mM, from about 7.5 mM to about 40 mM, from about 7.5 mM to about 50 mM, from about 10 mM to about 12.5 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 25 mM, from about 10 mM to about 30 mM, from about 10 mM to about 40 mM, or from about 10 mM to about 50 mM. In some embodiments, the first reagent (e.g., EDTA) can be present in the composition at about 1 mM, at about 2 mM, at about 3 mM, at about 4 mM, at about 5 mM, at about 7.5 mM, at about 10 mM, at about 12.5 mM, at about 15 mM, at about 20 mM, or at about 30 mM.

[0265] In some embodiments, the composition can comprise a second reagent. In some embodiments, the second reagent can solubilize the biomarker. In some embodiments, the second reagent can mitigate or reduce non-specific binding of the biomarker to a solid phase during the pre-processing or cleaning. In some embodiments, the solid phase can comprise a plate, a well, a transferring device (e.g., a pipette), or an interference capture particle (e.g., a Clean Bead). In some embodiments, the second reagent can comprise a non-ionic surfactant. In some embodiments, the second reagent can comprise polysorbate 20 (Tween-20). In some embodiments, the second reagent can comprise a polysorbate 20 analog. In some embodiments, the second reagent (e.g., Tween-20) can be present in the composition with a volume fraction (v / v) from about 0.01% to about 0.05%, from about 0.01% to about 0.1%, from about 0.01% to about 0.2%, from about 0.01% to about 0.3%, from about 0.01% to about 0.4%, from about 0.01% to about 0.5%, from about 0.01% to about 0.6%, from about 0.01% to about 0.7%, from about 0.01% to about 0.8%, from about 0.01% to about 0.9%, from about 0.01% to about 1%, from about 0.05% to about 0.1%, from about 0.05% to about 0.2%, from about 0.05% to about 0.3%, from about 0.05% to about 0.4%, from about 0.05% to about 0.5%, from about 0.05% to about 0.6%, from about 0.05% to about 0.7%, from about 0.05% to about 0.8%, from about 0.05% to about 0.9%, from about 0.05% to about 1%, from about 0.1% to about 0.2%, from about 0.1% to about 0.3%, from about 0.1% to about 0.4%, from about 0.1% to about 0.5%, from about 0.1% to about 0.6%, from about 0.1% to about 0.7%, from about 0.1% to about 0.8%, from about 0.1% to about 0.9%, from about 0.1% to about 1%, from about 0.2% to about 0.3%, from about 0.2% to about 0.4%, from about 0.2% to about 0.5%, from about 0.2% to about 0.6%, from about 0.2% to about 0.7%, from about 0.2% to about 0.8%, from about 0.2% to about 0.9%, from about 0.2% to about 1%, from about 0.3% to about 0.4%, from about 0.3%to about 0.5%, from about 0.3% to about 0.6%, from about 0.3% to about 0.7%, from about 0.3% to about 0.8%, from about 0.3% to about 0.9%, from about 0.3% to about 1%, from about 0.4% to about 0.5%, from about 0.4% to about 0.6%, from about 0.4% to about 0.7%, from about 0.4% to about 0.8%, from about 0.4% to about 0.9%, from about 0.4% to about 1%, from about 0.5% to about 0.6%, from about 0.5% to about 0.7%, from about 0.5% to about 0.8%, from about 0.5% to about 0.9%, or from about 0.5% to about 1%....

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of detecting a physiologically active target macromolecule comprising: a) providing a sample from a subject; and b) detecting a presence or absence of a biomarker for a disease in the sample, wherein the biomarker comprises: i) a physiologically active target macromolecule (PATM) or a portion or fragment thereof specific to a disease macromolecule pathology and the disease pathology, wherein the physiologically active target macromolecule comprises a macromolecule that is at least partially hyperphosphorylated, misfolded, post translationally modified, insoluble, truncated, mutated, present in an aggregated form, present in a tangle, or any combination thereof when compared to a non-physiologically active macromolecule (NPATM); ii) a conformation of the PATM or a portion or fragment thereof specific to a pathology of the macromolecule and the disease pathology; iii) a complex of PATM or a portion or fragment thereof and non- physiologically active target macromolecule (NPATM) or a portion or fragment thereof (PATM-NPATM complex); iv) a conformation of the PATM or a portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM-NPATM complex; v) a conformation of the NPATM or a portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM-NPATM complex; vi) a binding of NPATM or a portion or fragment thereof to PATM or a portion or fragment thereof; or vii) any combination of i) - vi).

2. The method of claim 1, wherein the PATM or the portion or fragment thereof comprises NPATM or a portion or fragment thereof that is at least partially hyperphosphorylated (HPT), mis-folded, post translationally modified, insoluble, truncated, mutated, present in an aggregated form, present in a tangle, or any combination thereof.WSGR Docket No. 64594-704.6013. The method of any one of the preceding claims, wherein the PATM or the portion or fragment thereof comprises a target protein or a portion or fragment thereof that comprises an ability to bind the NPATM or a portion or fragment thereof.

4. The method of any one of the preceding claims, wherein the NPATM or the portion or fragment thereof comprises a macromolecule or a portion or fragment thereof that is not associated with a disease state.

5. The method of claim 1, wherein the subject is pre-symptomatic for, or suspected of having, the disease and the method further comprises: detecting a presence of a disease pathology or a prognosis of the subject developing the disease when the biomarker for the disease is present in the sample; or detecting an absence of the disease pathology or a prognosis of the subject not developing the disease when the biomarker for the disease is absent in the sample.

6. The method of any one of the preceding claims, wherein the detecting comprises detecting the PATM-NPATM complex by detecting a binding of a capture moiety that is specific to: a) the PATM-NPATM complex, b) the conformation of the PATM or the portion or fragment thereof in the PATM- NPATM complex, c) the conformation of target macromolecule or a portion or fragment thereof in a PATM-NPATM complex, or d) a combination of a), b), and / or c).

7. The method of claim 6, wherein the capture moiety that is specific to the PATM- NPATM complex comprises an anti-hyperphosphorylated antibody against a specific phosphorylation site, an antibody against a conformation of the PATM or the portion or fragment thereof specific to target macromolecule pathology and disease pathology, an antibody against the PATM-NPATM complex, an antibody against a conformation of the PATM or the portion or fragment thereof when the PATM or the portion or fragment thereof is in the PATM- NPATM complex, NPATM or a portion or fragment thereof, an anti-PATM polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, a F(ab) antibody, a F(ab')2 antibody, chimeric antibody, a chimeric ’ab'2 antibody fragment, a molecular imprinted polymer (MIP), an aptamer, an alpaca nanobody, a llama derived nanobody, a portionWSGR Docket No. 64594-704.601 or fragment of any of these, or any combination thereof.

8. The method of claim 6 or claim 7, wherein the capture moiety comprises two or more capture moi eties.

9. The method of claim 7, wherein the recombinant antibody comprises a recombinantly tagged protein or a portion or fragment thereof.

10. The method of claim 9, wherein the recombinantly tagged protein or the portion or fragment thereof comprises an anti -tag antibody.

11. The method of claim 10, wherein the anti-tag antibody comprises an anti -recombinant tag antibody.

12. The method of claim 11, wherein the anti-recombinant tag comprises an anti-V5 tag, an anti-6His tag, an anti-8His tag, an anti-HA, an anti-FLAG, an anti-c-Myc, an anti-GST, an antifusion domain, or any combination thereof.

13. The method of claim 12, wherein the anti-V5 tag antibody comprises a monoclonal anti- V5 tag antibody.

14. The method of any one of the preceding claims, wherein the detecting comprises: capturing the PATM-NPATM complex; disrupting the PATM-NPATM complex to release the NPATM or the portion or fragment thereof; and detecting the NPATM or the portion or fragment thereof.

15. The method of any one of the preceding claims, wherein the detecting comprises: capturing the target macromolecule or the portion or fragment thereof on a substrate; and determining whether a captured target macromolecule or the portion or fragment thereof is PATM or a portion or fragment thereof by exposing it to a NPATM or a portion or fragment thereof, wherein the NPATM complexes with the captured target macromolecule when the captured target macromolecule is PATM.WSGR Docket No. 64594-704.60116. The method of any one of the preceding claims, wherein the NPATM or the portion or fragment thereof is labeled with a label.

17. The method of claim 16, wherein the label comprises a fluorophore, a stable isotope, a mass tag, a horse radish peroxidase (HRP), an alkaline phosphatase (ALP), a luciferase, a chemiluminescent substrate, an electrochemiluminescence substrate, a peptide, an oligonucleotide, a recombinant tag, an HA tag (YPYDVPDYA), a FLAG tag (DYKDDDDK), a c-Myc tag (EQKLISEEDL), a GST tag, a tandem mass tagged antibody, a tandem mass tagged peptide, a stable isotope labelled antibody or peptide, a fragment or portion of any of these, or any combination thereof.

18. The method of claim 17, wherein the chemiluminescent substrate comprises an isoluminol, a luminol, an acridinium ester, an AB El, or any combination thereof.

19. The method of claim 17, wherein the electrochemiluminescence substrate comprises a ruthenium.

20. The method of claim 17, wherein the peptide comprises an SmTrip9, an SmTriplO, or a combination thereof.

21. The method of any one of the preceding claims, wherein the detecting comprises detecting a conjugate or detection reagent in a sandwich immunoassay or an inhibition immunoassay.

22. The method of claim 21, wherein the conjugate comprises an antibody or capture moiety that directly or indirectly generates assay signal, fluorescence, HRP fluorescence, ALP fluorescence, light, bioluminescence, color, mass signature, immuno-PCR signal, or any combination thereof.

23. The method of claim 21 or claim 22, wherein the conjugate comprises an HRP, an ALP, a Luciferase, a fluorophore, a chemiluminescent substrate, an electrochemiluminescence substrate, a bioluminescent substrate, an oligonucleotide for immune-PCR, or any combination thereof.

24. The method of claim 23, comprising the chemiluminescent substrate, wherein theWSGR Docket No. 64594-704.601 chemiluminescent substrate comprises an isoluminol, a luminol, an acridinium ester, an ABEI, a HRP substrate, an ALP substrate, an immuno-PCR oligonucleotide labeled antibody or peptide, stable isotope labelled antibodies or peptides, peptides labeled with light isotopes, tandem mass tagged antibodies or peptides, a fragment or portion of any of these, or any combination thereof.

25. The method of claim 23, comprising the chemiluminescence substrate, wherein the electrochemiluminescence substrate comprises ruthenium.

26. The method of claim 23, comprising the bioluminescent substrate, wherein the bioluminescent substrate comprises SmTrip9 or SmTriplO.

27. The method of any one of the previous claims, wherein an anti-PATM capture moiety is labeled with SmTrip9 and an anti-PATM-NPATM capture moiety is labeled with SmTriplO.

28. The method of any one of the previous claims, wherein an anti-PATM capture moiety is labeled with SmTriplO and an anti-PATM-NPATM capture moiety is labeled with SmTrip9.

29. The method of any one of the preceding claims, wherein the detecting of the biomarker comprises detecting a specific phosphorylated epitope of the Target macromolecule.

30. The method of any one the preceding claims, further comprising measuring an assay signal, dose, or PAT or PATM-NPATM concentration.

31. The method of any one of the preceding claims, wherein the detecting of the biomarker comprises detecting through electrophoresis, immunoblotting, immunoprecipitation, autoradiography, mass spectrometry, proteomics, protein separation, western blotting, protein identification, immunoassay, light scattering, spectrometry, calorimetry, biolayer interferometry (BLI), surface plasmon resonance (SPR), or other label-free optical techniques for measuring biomolecular interactions, or any combination thereof.

32. The method of any one of the preceding claims, wherein the detecting of the biomarker comprises detecting through MS, time-of-flight mass spectrometry (TOF-MS), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), LC-MS, or LC-MS / MS.

33. The method of any one of the preceding claims, wherein the detecting of the biomarkerWSGR Docket No. 64594-704.601 comprises binding the biomarker to biomarker capture particles comprising a biomarker capture moiety.

34. The method of claim 33, wherein the method further comprises quantitating the amount of biomarker that binds to the biomarker capture particles.

35. The method of claim 33 or claim 34, wherein the biomarker capture particles are coated with at least a first type of antibody specific to the first type of epitope and a second type of antibody specific to the second type of epitope.

36. The method of any one of claims 33-35, wherein the biomarker capture particles comprise agglutination particles, wherein the agglutination particles aggregate upon binding to the biomarker.

37. The method of any one of claims 33-36, wherein the biomarker capture particles comprise microparticles.

38. The method of any one of claims 33-37, wherein the biomarker capture particles comprise beads.

39. The method of any one of claims 33-38, wherein the biomarker capture particles comprise a metal.

40. The method of any one of claims 33-39, wherein the biomarker capture particles are magnetic.

41. The method of any one of claims 33-40, wherein the biomarker capture particles comprise: a plurality of magnetic beads, and a plurality of non-magnetic beads, wherein the plurality of magnetic beads is larger in size than the plurality of nonmagnetic beads, and wherein one or more of the pluralities of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker.

42. The method of claim 41, wherein a concentration of the plurality of non-magnetic beadsWSGR Docket No. 64594-704.601 decreases upon removal of the complex.

43. The method of claim 33, wherein the biomarker capture moiety comprises an antibody or antibody binding fragment.

44. The method of claim 43, wherein the antibody or antibody binding fragment comprises an autoantibody, a therapeutic antibody, an immunoglobulin class, an immunoglobulin subclass, a circulating antibody, a secretory antibody, an alpaca derived nanobody, an animal derived antibody, a fragment of any of these, or any combination thereof.

45. The method of any one of the preceding claims, wherein the sample comprises a biological fluid sample.

46. The method of claim 45, wherein the biological fluid sample comprises a blood, a plasma, a serum, a reconstituted dry blood spot, a capillary collection, a saliva, a saline oral rinse, a stool, a urine, tears, a cerebrospinal fluid, or any combination thereof.

47. The method of any one of the preceding claims, further comprising removing interferences from the sample prior to the detecting of the presence or absence of the biomarker.

48. The method of claim 47, wherein the removing interferences from the sample comprises cleaning the sample by removing the interferences from the sample using interference capture particles.

49. The method of claim 48, wherein the interference capture particles comprise an interference capture moiety that interacts with a sample interference.

50. The method of any one of claims 47-49, wherein the interferences comprise a human anti-mouse antibody (HAMA), a rheumatoid factor (RF), an autoantibody to a Target macromolecule or a pTarget macromolecule, a Macro-Target macromolecule complex of a Target macromolecule, a pTarget macromolecule, both a Target macromolecule and a pTarget macromolecule with autoantibodies, an anti-streptavidin interference, an anti-biotin interference, an anti-PEG interference (or PEOn), or any combination thereof.

51. The method of any one of the preceding claims, wherein the subject is a human.WSGR Docket No. 64594-704.60152. The method of claim 1, further comprising cross-linking the PATM-NPATM complex using a cross-link agent.

53. The method of claim 52, wherein the cross-link agent comprises glutaraldehyde or formaldehyde.

54. The method of any one of the preceding claims, further comprising binding the biomarker with a detection antibody.

55. The method of claim 54, further comprising measuring the binding of the biomarker with the detection antibody.

56. The method of claim 55, further comprising comparing the binding of the biomarker with the detection antibody to a standard curve.

57. The method of claim 56, wherein the standard curve is generated from biomarker capture particles bound to known amounts of the biomarker.

58. The method of claim 57, wherein the biomarker capture particles are coated with an antibody, and wherein the standard curve is generated from known amounts of liquid or lyophilized peptide that is added to the biomarker capture particles.

59. The method of any one of the preceding claims, further comprising quantitating the biomarker.

60. The method of any one of the preceding claims, wherein the sample comprises a first sample and the method further comprises monitoring the biomarker over time to determine an increase or decrease in an amount of the biomarker in a second sample of the subject, relative to the amount of biomarker in the first sample.

61. The method of any one of the preceding claims, wherein the method comprises diagnosing the subject with the disease based at least in part on detecting: i) the PATM or a fragment or portion thereof specific to target macromolecule pathology and disease pathology; ii) the conformation of the PATM or a fragment or portion thereofWSGR Docket No. 64594-704.601 specific to target macromolecule pathology and disease pathology; iii) the complex of PATM or a fragment or portion thereof and non- physiologically active target macromolecule or a fragment or portion thereof (PATM-NPATM complex); iv) the conformation of the NPATM or a fragment or portion thereof when the NPATM or the fragment or portion thereof is in the PATM-NPATM complex; v) the conformation of the PATM or a fragment or portion thereof when the PATM or the fragment or portion thereof is in the PATM-NPATM complex; vi) the binding of NPATM or a fragment or portion thereof to PATM or a fragment or portion thereof; or vii) any combination of i) - vi).

62. The method of claim 61, further comprising administering a treatment to the subject for the disease.

63. The method of claim 61 or claim 62, wherein the target macromolecule pathology comprises an insoluble target macromolecule aggregate, a neurofibrillary tangle, a PATM or a fragment or portion thereof, a PATM-NPATM complex, or any combination thereof.

64. The method of any one of claims 61-63, further comprising determining a course of a disease progression based on the quantity of the biomarker detected, the quantity of the amount of target macromolecule or the fragment or portion thereof that is physiologically active, or a combination thereof.

65. The method of any one of the preceding claims, wherein a signal to noise ratio of the detection is more than about 3.0.

66. The method of any one of the preceding claims, wherein the biomarker comprises a pTau231.

67. The method of any one of the preceding claims, wherein the biomarker comprises a pTau217.WSGR Docket No. 64594-704.60168. The method of any one of the preceding claims, wherein the biomarker comprises a pTau231 and pTau217.

69. The method of any one of the preceding claims, wherein the macromolecule comprises a human prion protein (PrP), a tau protein, a transactive response DNA binding protein 43 (TDP- 43), an M protein, an alpha-synuclein (a-syn), a glial fibrillary acidic protein (GFAP), a betaamyloid 1-42 (AB (1-42)), a neurofilament light chain, a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, an HGF protein, a CTSD protein, a amylin, an Islet Amyloid Polypeptide (IAPP), a huntingtin (HTT) protein, a p53 protein, a tumor suppressor protein, an oncoprotein, an asprosin protein, a suprabasin protein, a cyclooxy genase- 2 (COX-2) protein, an ezrin protein, a portion or fragment of any of these, or any combination thereof.

70. The method of any one of the preceding claims, wherein the disease comprises a Creutzfeldt-Jakob disease, an Amyotrophic Lateral Sclerosis, a Multiple Myeloma, a Parkinson's Disease, a Type II diabetes, a Huntington's disease, a cancer, a Basal cell carcinoma, an Alzheimer’s disease, or any combination thereof.

71. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a human prion protein (PrP), a tau protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active human prion protein (PrP), a physiologically active tau protein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active human prion protein (PrP), a non- physiologically active tau protein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Creutzfeldt-Jakob disease.

72. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a transactive response DNA binding protein 43 (TDP-43), a portion or fragment thereof, or any combination thereof; b) the PATM comprises a physiologically active TDP-43, a portion or fragment thereof, or any combination thereof;WSGR Docket No. 64594-704.601 c) the NPATM comprises a non-physiologically active TDP-43, a portion or fragment thereof, or any combination thereof; and d) the disease comprises an Amyotrophic Lateral Sclerosis (ALS).

73. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises an M protein, a portion or fragment thereof, or any combination thereof; b) the PATM comprises a physiologically active M protein, a portion or fragment thereof, or any combination thereof; c) the NPATM comprises a non-physiologically active M protein, a portion or fragment thereof, or any combination thereof; and d) the disease comprises a Multiple Myeloma.

74. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises an alpha-synuclein (a-syn), a glial fibrillary acidic protein (GFAP), a beta-amyloid 1-42 (AB (1-42)), a tau, a neurofilament light chain, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active alpha-synuclein (a-syn), a physiologically active glial fibrillary acidic protein (GFAP), a physiologically active betaamyloid 1-42 (AB (1-42)), a physiologically active tau, a physiologically active neurofilament light chain, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active alpha-synuclein (a-syn), a non- physiologically active glial fibrillary acidic protein (GFAP), a non-physiologically active betaamyloid 1-42 (AB (1-42)), a non-physiologically active tau, a non-physiologically active neurofilament light chain, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Parkinson’s disease.

75. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a follistatin protein, an insulin-like growth factor binding protein 2, a FABP4 protein, an HGF protein, a CTSD protein, an amylin, an Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active follistatin protein, a physiologically active insulin-like growth factor binding protein 2, a physiologically active FABP4 protein, a physiologically active HGF protein, a physiologically active CTSD protein, a physiologicallyWSGR Docket No. 64594-704.601 active amylin, a physiologically active Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active follistatin protein, a non- physiologically active insulin-like growth factor binding protein 2, a non-physiologically active FABP4 protein, a non-physiologically active HGF protein, a non-physiologically active CTSD protein, a non-physiologically active amylin, a non-physiologically active Islet Amyloid Polypeptide (IAPP), a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a diabetes.

76. The method of claim 75, wherein the diabetes comprises a Type II diabetes.

77. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a glial fibrillary acidic protein, a beta-amyloid 1- 42 (AB (1-42)), a tau protein, huntingtin (HTT) protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active p53 protein, a physiologically active tumor suppressor protein, a physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active tumor suppressor protein, a non- physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a Huntington's disease.

78. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a p53 protein, a tumor suppressor protein, an oncoprotein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active p53 protein, a physiologically active tumor suppressor protein, a physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active p53 protein, a non- physiologically active tumor suppressor protein, a non-physiologically active oncoprotein, a portion or fragment of any of these, or any combination thereof; and d) the disease comprises a cancer.

79. The method of any one of the preceding claims, wherein:WSGR Docket No. 64594-704.601 a) the target macromolecule comprises an asprosin protein, a suprabasin protein, a cyclooxygenase-2 (COX-2) protein, an ezrin protein, a portion or fragment of any of these, or any combination thereof; b) the PATM comprises a physiologically active asprosin protein, a physiologically active suprabasin protein, a physiologically active cyclooxygenase-2 (COX-2) protein, a physiologically active ezrin protein, a portion or fragment of any of these, or any combination thereof; c) the NPATM comprises a non-physiologically active asprosin protein, a non- physiologically active suprabasin protein, a non-physiologically active cyclooxygenase-2 (COX- 2) protein, a non-physiologically active ezrin protein, a portion or fragment of any of these, or any combination thereof, and; d) the disease comprises a basal cell carcinoma.

80. The method of any one of the preceding claims, wherein: a) the target macromolecule comprises a Tau protein or a portion or fragment thereof; b) the PATM comprises a physiologically active Tau (PAT) protein or a portion or fragment thereof; c) the NPATM comprises a non-physiologically active Tau (NPAT) protein or a portion or fragment thereof, and; d) the disease comprises an Alzheimer’s disease.

81. The method of claim 80, wherein the PAT protein or the portion or fragment thereof comprises pTaul81, pTaul99, pTau202, pTau205, pTau212, pTau214, pTau217, pTau231, pTau235, pTau262, pTau396, pTau404 or pTau422, or any combination thereof.

82. The method of claim 81, wherein the PAT protein or the portion or fragment thereof comprises a pTau231.

83. The method of claim 81 or claim 82, wherein the PAT protein or the portion or fragment thereof comprises a microtubule-binding region (MTBR) of tau comprising:(a) MTBR R1 residues 244-274, MTBR R2 residues 275-305, MTBR R3 residues 306-336, MTBR R4 residues, or any combination thereof;(b) MTBR sites implicated in tau aggregation comprising residues 275-280 (vqiink), 306-311 (vqivyk), or a combination thereof;(c) residues 225-242 near residue 243 and R1 ;WSGR Docket No. 64594-704.601(d) residues 226-264 near residue 260 within R1 ;(e) R1 residues 256-273 (vkskigstenlkhqpggg), R2 residues 287-304 (vqskcgskdnikhvppgg), R3 residues 318-335 (vtskcgslgnihhkpggg), R4 residues 350-364 (vqskigsldnithvpggg), and combinations thereof; or(f) R1 residues 268-271 (hqpg), R2 residues 299-302 (hvpg), R3 residues 330-333 (hkpg), R4 residues 362-365 (hvpg), or any combination thereof.

84. The method of any one of claims 81-83, wherein the PAT protein or the portion or fragment thereof comprises a specific phosphorylation site at Thrl81, Seri 99, Ser202, Thr205, Thr212, Ser214, Thr217, Thr231, Ser235, Ser262, Ser396, Ser404, Ser422, or any combination thereof.

85. The method of any one of claims 81-84, wherein the non-PAT protein or the portion or fragment thereof comprises recombinant human Tau-441, or a fragment or portion of tau441 peptide selected from a full length peptide, a truncated peptide, a mutated peptide, a modified recombinant amino acid sequence in any position(s) in the peptide, amino acid modifications (e.g., phosphorylation, methylation, radioisotopes, oligonucleotides, fluorophore, etc.) in any position(s) in the peptide, a peptide comprising one or more of the MTBR regions such as Rl, or R2, or R3, or R1-R2, or R2-R3, or R1-R3, or R2-R4, or R3-R4, or R1-R4, or any combination thereof, or a peptide comprising a tau aggregation sequence at the begging of R2 (VQIINK tau aggregation site) and / or R3 (VQIVYK tau aggregation site), a portion or fragment of any of these, or any combination thereof.

86. The method of any one of claims 81-85, wherein the detecting of the biomarker comprises detecting a specific phosphorylated epitope of Tau.

87. The method of any one of claims 81-86, wherein the specific phosphorylated epitope of Tau comprises anti-Thrl81, anti-Serl99, anti-Ser202, anti-Thr205, anti-Thr212, anti-Ser214, anti-Thr217, anti-Thr231, anti-Ser235, anti-Ser262, anti-Ser396, anti-Ser404, or anti-Ser422, and the conjugate is against a different phosphorylated or non-phosphorylated Tau epitope.

88. The method of claim 44, wherein the antibody or antibody binding fragment comprises a tau-441 peptide, or a fragment or portion of tau441 peptide comprising at least one of MTBR R1-R4.WSGR Docket No. 64594-704.60189. The method of claim 62, wherein the treatment comprises a medicament or disease modifying therapy (DMT) that targets amyloid pathology (amyloid plaque), tau pathology, neuroregeneration, cognitive function, or an anti-sense oligonucleotide (ASO), or a pharmaceutically effective amount of TRIM21, or any combination thereof.

90. The method of claim 89, wherein the tau pathology comprises an insoluble tau aggregate, a neurofibrillary tangle, a PAT protein or a fragment or portion thereof, a PAT-Tau complex, or any combination thereof.

91. The method of claim 89, wherein the medicament comprises a galantamine, a rivastigmine, a donepezil, an immunotherapy, or any combination thereof.

92. The method of any one of claims 6-8, wherein the capture moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3).

93. The method of claim 92, wherein the VH comprises a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5).

94. The method of claim 92 or claim 93, wherein the VH comprises a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4).

95. The method of any one of claims 92-94, wherein the VL comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9).

96. The method of claim 95, wherein the VL comprises a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8).

97. The method of any one of claims 92-96, wherein the VL comprises a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

98. The method of any one of claims 6-8 and 92-97, wherein the capture moiety comprises: a CDR-H3 comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3);WSGR Docket No. 64594-704.601 a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5); a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4); a CDR-L3 comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9); a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8); and a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

99. The method of any one of claims 92-98, wherein the VH comprises a sequence with at least 80% sequence identity to the sequenceQVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS T YNEKFKSIGA.TLTVDTS S STAYMQLS SLTSEDS AVYYC ARDDGYS AWF AYWGQGTL VTVSA (SEQ ID NO: 10).

100. The method of any one of claims 92-99, wherein the VL comprises a sequence with at least 80% sequence identity to the sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11).

101. The method of any one of claims 92-100, wherein the VH comprises the amino acid sequence of QVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10).

102. The method of any one of claims 92-101, wherein the VL comprises the amino acid sequence ofDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11).

103. The method of claim 43 or 44, wherein the antibody or antibody binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3).WSGR Docket No. 64594-704.601104. The method of claim 103, wherein the VH comprises a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5).

105. The method of claim 103 or claim 104, wherein the VH comprises a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4).

106. The method of any one of claims 103-105, wherein the VL comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9).

107. The method of claim 106, wherein the VL comprises a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8).

108. The method of claim 106 or claim 107, wherein the VL comprises a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

109. The method of any one of claims 43, 44, and 103-108, wherein the antibody or antibody binding fragment comprises: a CDR-H3 comprising the amino acid sequence of DDGYSAWFAY (SEQ ID NO: 3); a CDR-H2 comprising the amino acid sequence of YPGSDS (SEQ ID NO: 5); a CDR-H1 comprising the amino acid sequence of GYTFTSH (SEQ ID NO: 4); a CDR-L3 comprising the amino acid sequence of FQGSHVPFT (SEQ ID NO: 9); a CDR-L2 comprising the amino acid sequence of KVFNRFS (SEQ ID NO: 8); and a CDR-L1 comprising the amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

110. The method of any one of claims 103-109, wherein the VH comprises a sequence with at least 80% sequence identity to the sequenceQVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10).

111. The method of any one of claims 103-110, wherein the VL comprises a sequence with at least 80% sequence identity to the sequenceDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRFWSGR Docket No. 64594-704.601SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11).

112. The method of any one of claims 103-111, wherein the VH comprises the amino acid sequence ofQVQLQQPGAELVKPGTSVKMSCKASGYTFTSHWMHWVKQRPGQGLEWIGDIYPGSDS TNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARDDGYSAWFAYWGQGTL VTVSA (SEQ ID NO: 10).

113. The method of any one of claims 103-112, wherein the VL comprises the amino acid sequence ofDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 11).

114. The method of any one of the previous claims, wherein the detecting comprises:(a) contacting the sample comprising the biomarker with a composition comprising a first reagent and a second reagent, wherein the first reagent deactivates one or more interference enzymes, wherein the second reagent solubilizes the biomarker; and(b) contacting the sample with a plurality of particles, wherein the plurality of particles comprises a capture moiety to bind the biomarker.

115. A method, comprising:(a) contacting a sample comprising a biomarker with a composition comprising a first reagent and a second reagent, wherein the first reagent deactivates one or more interference enzymes, wherein the second reagent solubilizes the biomarker; and(b) contacting the sample with a plurality of particles, wherein the plurality of particles comprises a capture moiety to bind the biomarker.

116. The method of claim 114 or 115, further comprising, prior to (b), contacting the sample with an additional plurality of particles, wherein the additional plurality of particles binds one or more interference molecules in the sample.WSGR Docket No. 64594-704.601117. The method of claim 114 or 115, wherein the one or more interference enzymes comprise one or more endogenous phosphatase.

118. The method of claim 114 or 115, wherein the second reagent further reduces nonspecific binding of the biomarker to the additional plurality of particles.

119. The method of claim 114 or 115, wherein the composition further comprises a third reagent, wherein the third reagent blocks one or more biotin binding moieties.

120. The method of any one of the previous claims, wherein the detecting comprises: contacting a sample comprising one or more phosphorylated tau proteins with a composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein.

121. A method, comprising: contacting a sample comprising one or more phosphorylated tau proteins with a composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein.

122. The method of any one of the preceding claims, wherein the detecting comprises detecting a PATM in a sample, comprising:(a) capturing the PATM with a capture moiety to form a capture rnoiety-PATM complex;(b) contacting the capture moiety' -PATM complex with a non-PAT (NPATM) or a portion or fragment thereof, to form a capture moiety -PATM-NP ATM complex;(c) contacting the capture moiety-HPT-PATM-NPATM complex with a detection moiety, wherein the detection moiety binds to the capture moiety -PATM-NP ATM complex to form a capture moiety-HPT-PATM-NPATM complex-detection moiety complex; and(d) detecting the capture moiety-HPT-PATM-NPATM complex-detection moiety complex.WSGR Docket No. 64594-704.601123. A method of detecting a hyperphosphory lated tau (HPT) protein in a sample, comprising:(a) capturing the HPT protein with a capture moiety to form a capture moiety-HPT complex;(b) contacting the capture moiety’ -HPT complex with a non-hyperphosphorylated tau (nTau) protein or a portion or fragment thereof, to form a capture moiety-HPT-nTau complex;(c) contacting the capture moiety-HPT-nTau complex with a detection moiety, wherein the detection moiety binds to the capture moiety-HPT-nTau complex to form a capture moiety- HPT-nTau complex-detection moiety complex; and(d) detecting the capture moiety-HPT-nTau complex-detection moiety complex.

124. A composition, comprising:(a) a first reagent configured to deactivate one or more interference enzymes;(b) a second reagent configured to solubilize a biomarker;(c) a third reagent configured to block biotin binding moieties; or(d) any combination of (a)-(c).

125. A kit, comprising:(a) a first reagent configured to deactivate one or more interference enzymes;(b) a second reagent configured to solubilize a biomarker;(c) a third reagent configured to block biotin binding moieties; or(d) any combination of (a)-(c).

126. The kit of claim 125, further comprising Tris-HCl, NaCl, or sodium azide, or any combination thereof.

127. A composition comprising a first population of particles and a second population of particles, wherein a particle of the first population of particles comprises a first antibody that is specific to at least a portion of a first phosphorylated tau protein, wherein a particle of the second population of particles comprises a second antibody that is specific to at least a portion of a second phosphorylated tau protein.

128. A composition comprising a monoclonal antibody that is specific to a PATM-NPATM complex.

129. A composition comprising a monoclonal antibody that is specific to the conformation ofWSGR Docket No. 64594-704.601PATM or a fragment or portion thereof in a PATM-NPATM complex.

130. The composition of claim 128 or claim 129, wherein the monoclonal antibody is a human monoclonal antibody, an animal antibody, or a humanized animal antibody.

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