Phosphorylated TAU immunoassays and methods of producing and using same

Abstract

Compositions, kits, systems, and methods for determining the presence and / or concentration of a phosphorylated Tau biomarker (such as, but not limited to, pTau217) in a biological sample and / or determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual are disclosed. The compositions, kits, systems, and methods utilize at least two means for binding to Tau, including one means that is capable of distinguishing the phosphorylated Tau biomarker from unphosphorylated Tau, so that at least one sandwich complex can be formed. The methods include determining an amount of pTau present in the biological sample based on the amount of the sandwich immunocomplex formed.

Description

PHOSPHORYLATED TAU IMMUNOASSAYS ANDMETHODS OF PRODUCING AND USING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 779,632, filed March 28, 2025; and US Provisional Application No.63 / 879,315, filed September 10, 2025. The entire contents of each of the above-referenced patent applications are hereby expressly incorporated herein by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via WIPO-Sequence in computer readable form as an XML file. The Sequence Listing, created March 24, 2026, is named "2024P1911WO_SequenceListing.xml" and is 14,918 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference.BACKGROUND

[0003] Certain immunoassays detect a complex between a biomarker-of-interest, a first biomarker-binding antibody, and a second biomarker-binding antibody that is labeled. In an example of a sandwich immunoassay format, a first, biomarker-binding antibody, such as a capture antibody suitable for binding a biomarker-of-interest, is attached to an insoluble material or substrate to form a solid phase reagent. A light reagent including a second antibody that also binds the biomarker-of-interest at an epitope that does not substantially overlap with the epitope to which the first antibody binds, such as a labeling antibody including a signal moiety, is also provided. When contacted with a biological sample under predetermined conditions and including a predetermined antigen or biomarker-of-interest, the solid phase reagent, biomarker-of-interest, and light reagent form a complex capable of generating a detectable signal that indicates the presence and / or amount of the biomarker- of-interest.

[0004] Biomarkers act as surrogates for clinically meaningful outcomes and may or may not reflect the pathogenesis underlying a disease. Examples of clinical utility include diagnosis, the prediction of disease progression or regression, and prognostication ofmortality. A biomarker should be easily acquired, reliably measured, arid available for serial monitoring. Ideally, a biomarker would also provide an advantage of currently used clinical measures in ease, timeframe, and / or expense.

[0005] Early detection of Alzheimer's disease and differentiation from other neuropathies before severe symptoms manifest is a critical clinical need. Commonly used methods for detection include PET images, cognitive tests, and a set of blood-based assays including pTau-181, aB40 / 42 ratio, and others. However, these methods lack sensitivity, are expensive, and are often required to be used together for specificity.

[0006] Therefore, there is a need in the art for new and improved immunoassays for detection of Alzheimer's disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments.

[0008] FIG. 1 depicts one non-limiting embodiment of a sandwich immunoassay constructed in accordance with the present disclosure and utilizing a capture monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to pTau217 and is biotinylated, a detection mAb or antigen-binding fragment thereof that specifically binds to Tau and is labeled with an acridinium ester, and a streptavidin-coated magnetic particle solid phase reagent.

[0009] FIG. 2 depicts an exemplary block diagram of one non-limiting embodiment of a computer system 1100 configured for use in accordance with the present disclosure.

[0010] FIG. 3 depicts a structure of one non-limiting embodiment of a biotin that can be utilized in accordance with the present disclosure.

[0011] FIG. 4 depicts structures of non-limiting embodiments of acridinium esters that can be utilized in accordance with the present disclosure.

[0012] FIG. 5 graphically depicts an RLU chromatogram of an NHS-TSPAE labeled anti-Tau detection antibody.

[0013] FIG. 6 graphically depicts a signal in function of pTau217 concentrations on the Atellica® IM analyzer (Siemens Healthineers USA, Malvern, PA) using HL10 pTau217 as capture antibody and ADx204 as detection antibody, with synthetic phosphorylated Tau peptide as the sample.

[0014] FIG. 7 graphically depicts a box and whisker plot demonstrating the ability to distinguish between Alzheimer's disease (AD) and non-AD and normal / healthy patients using the immunoassay of FIG. 6.

[0015] FIG. 8 graphically depicts a curve response using RD-085 pTau217 as capture antibody and ADx204 as detection antibody, with synthetic phosphorylated Tau peptide as the sample.

[0016] FIG. 9 graphically depicts a curve response using 10H6 pTau217 as capture antibody and ADx204 or TauJ.5H3 as detection antibody, with synthetic phosphorylated Tau peptide GSK3beta as the sample.

[0017] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. The figures are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0018] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques andprocedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0020] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0021] All of the compositions, devices, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, devices, kits, and / or methods have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, devices, kits, and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0022] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0023] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherently present, therein.

[0024] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0025] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least, one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Similarly, the term "at least two" will be understood to include two as well as any quantity more than two, including but not limited to, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.

[0026] The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and, unless explicitly stated otherwise, is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0027] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition " A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0028] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0029] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0030] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the present disclosure. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value.

[0031] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, " A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0032] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time,or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item,

[0033] As used herein, the phrase "associated with" includes both direct association of two moieties to one another as well as indirect association of two moieties to one another. Non-limiting examples of associations include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety.

[0034] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), recombinant antibodies, synthetic antibodies, chimeric antibodies, and conjugates of any of the above, so long as they exhibit the desired biological activity of analyte binding. The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, IgG 2, lgG3, lgG4, IgAl, and lgA2).

[0035] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Thus, the term "antigen-binding fragment" or "antigen-binding portion" refers to any fragment of a full-length immunoglobulin (Ig) molecule (i.e., an intact antibody) that competes with the intact antibody for specific antigen binding. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, singlechain antibodies, single domain antibodies (such as but not limited to, nanobodies), isolated CDR-H3, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody. Antigen-binding fragments utilized in accordance with the present disclosure can be obtained commercially and / or produced using conventional recombinant and / or enzymatic techniques and are screened for antigen binding properties in the same manner as intact antibodies.

[0036] " Fv" is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This fragment consists of a dimer of one heavy- and one light¬ chain variable region domain in tight, non -covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of a Fv including only three complementarity determining regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present, in all antibody molecules in their naturally occurring conformations. K and A light chains refer to the two major antibody light chain isotypes.

[0037] By the term, "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0038] The term "antigen" as used herein is defined as a molecule that provokes an immune response, which may involve either antibody production, or the activation of specific immunologically competent cells, or both. Antigens may include any macromolecule, including virtually all proteins or peptides. Further, an antigen can be generated, synthesized or derived from a biological sample including a tissue sample, a tumor sample, a cell or a biological fluid.

[0039] The term "immunoglobulin" or " Ig," refers to a class of proteins, which function as antibodies. The five members included in this class of proteins are IgA, IgG, IgIVI, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulinproduced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to the allergen.

[0040] By "isolated" is meant a material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.

[0041] The terms ''polypeptide,” "polypeptide fragment," "peptide," and "protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides, or "enzymes," which typically catalyze (i.e., increase the rate of) various chemical reactions.

[0042] The recitation polypeptide "variant" refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain non-limiting embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain non-limiting embodiments, the polypeptide variant comprises conservative substitutions (such as 1, 2, 3, 4, 5, or 1-5 substitutions) and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced with different amino acid residues.

[0043] The term "biomarker" or "biological marker" is used herein, consistent with its use in the art, to refer to an entity whose presence, level, or form, correlates with a particular biological event or state of interest, so that it is considered to be a "marker" of that event orstate. To give but a few examples, in some non-limiting embodiments, a biomarker may be or include a marker for a particular disease state, or for a likelihood that, a particular disease, disorder, or condition may develop, occur, or reoccur. In some non-limiting embodiments, a biomarker may be or include a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus, in some non-limiting embodiments, a biomarker is predictive, prognostic, and / or diagnostic of the relevant biological event or state of interest. In some non-limiting embodiments, a biomarker is a possible biomarker of the relevant biological event or state of interest. A biomarker may be an entity of any chemical class. For example, in some non-limiting embodiments, a biomarker may be or include a nucleic acid, a polypeptide, a small molecule, or a combination thereof. In some non-limiting embodiments, a biomarker is a cell surface marker. In some non-limiting embodiments, a biomarker is intracellular. In some non-limiting embodiments, a biomarker is found in a particular tissue (e.g., lung tissue). In some non-limiting embodiments, a biomarker is found outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0044] As described herein, in some non-limiting embodiments, the biomarker detected is a phosphorylated Tau (pTau) biomarker, which serves as a biological marker for Alzheimer's disease and, in particular (but non-limiting) embodiments, also serves to distinguish AD from other neuropathic diseases and disorders. In some non-limiting embodiments, the pTau biomarker is a Tau protein or characteristic fragment thereof that is phosphorylated at a specific residue thereof. Non-limiting examples thereof include: pTaul81, which is phosphorylated at the threonine at position 181 of the full-length human Tau protein; pTau212, which is phosphorylated at the threonine at position 212 of the full-length human Tau protein; pTau214, which is phosphorylated at the threonine at position 214 of the full- length human Tau protein; pTau217, which is phosphorylated at the threonine at position 217 of the full-length human Tau protein; pTau220, which is phosphorylated at the threonine at position 220 of the full-length human Tau protein; and pTau2.31, which is phosphorylated at the threonine at position 231 of the full-length human Tau protein. In certain non-limiting embodiments, the pTau Biomarkers include variants and characteristic fragments of pTau including proteins and peptides having an amino acid sequence that has at least 90%, 95%, 99%, or 100% sequence identity to at least a portion of the wild type Tau (SEQ ID NO:11), so long as the Tau variant is phosphorylated at a specific position thereof.SEQ ID NO:11.

[0045] The term "characteristic fragment" refers to a fragment of a biomarker (e.g., pTau Biomarker) that is sufficient to identify the biomarker from which the fragment was derived. For example, in some non-limiting embodiments, a "characteristic fragment" of a biomarker is one that contains an amino acid sequence or a collection of amino acid sequences that together allow for the biomarker from which the fragment was derived to be distinguished from other possible biomarkers, proteins, or polypeptides. In some non-limiting embodiments, a characteristic fragment includes at least 10, at least 20, at least 30, at least 40, or at least 50 amino acids. In certain non-limiting embodiments, a "pTau characteristic fragment" refers to a fragment of a biomarker that has at least 90%, at least 95%, or at least 99% sequence identity to a portion of SEQ ID NO:11 and contains the specific residue for which phosphorylation is being determined (for example, but not by way of limitation, a characteristic fragment of pTau217 contains the Thr 217 residue that is phosphorylated).

[0046] In addition, the pTau biomarker must contain two epitopes: a first epitope that is different from and substantially non-overlapping with a second epitope containing the phosphorylated residue, whereby a first antibody can bind to a first, Tau specific epitope at the same time that a second antibody can bind to a second, pTau specific epitope so that a sandwich complex can be formed.

[0047] The term "detection agent" as used herein refers to any element, molecule, functional group, compound, fragment, or moiety that is detectable. In some non-limiting embodiments, a detection agent is provided or utilized alone. In some non-limiting embodiments, a detection agent is provided and / or utilized in association with (e.g., joined to) another agent. Examples of detection agents include, but are not limited to: various ligands, radionuclides (e.g.,3H,14C,18F,19F,32P,35S,135I,12-’l,123I,64Cu,187Re,111In,90Y,99mTc,177Lu,89Zr etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridiniumesters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available. In certain non-limiting embodiments, a detection agent suitable for use herein includes acridinium esters such as TSPAE (such as, but not limited to, TSPAE-TEG-MCC and TSPA-NHS), HEGAE (as described in U. S. Patent No. 6,664,043), DMAE, and the like.

[0048] As used herein, "diagnostic test" is a step or series of steps that is or has been performed to attain information that is useful in determining whether a patient has a disease, disorder, or condition and / or in classifying a disease, disorder, or condition into a phenotypic category or any category having significance with regard to prognosis of a disease, disorder, or condition, or likely response to treatment (either treatment in general or any particular treatment) of a disease, disorder, or condition. Similarly, "diagnosis" refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder, or condition, state, staging, or characteristic of a disease, disorder, or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis, and / or information useful in selecting an appropriate treatment or additional diagnostic testing. Selection of treatment may include the choice of a particular therapeutic agent or other treatment modality such as surgery, radiation, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc. Selection of additional diagnostic testing may include more specific testing for a given disease, disorder, or condition.

[0049] The term "biological fluid sample" as used herein will be understood to include any liquid test sample that may be obtained from a patient and utilized in accordance with the present disclosure. Examples of biological fluid samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, combinations thereof, and the like.

[0050] As used herein, the term "volume" as it relates to the liquid test samples utilized in accordance with the present disclosure typically refers to a volume of liquid test sample in a range of from about 0.1 μl to about 100 μl, or a range of from about 1 μl to about 75 μl, or a range of from about 2 μl to about 60 μl, or a value less than or equal to about 50 μl, or the like.

[0051] The term "patient" as utilized herein includes human and veterinary subjects. In certain non-limiting embodiments, a patient is a mammal. In certain other non-limiting embodiments, the patient is a human. The term "mammal" for purposes of diagnosis / treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

[0052] A "health care provider" or "health care decision maker" includes any individual authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient. In the context of identifying useful new drugs to treat lung disease, a health care provider can be an individual who is not authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient.

[0053] The term "means for binding" or "specific binding partner" (as used in particular (but not byway of limitation) herein in the term "target analyte-specific binding partner") will be understood to refer to any molecule capable of specifically associating with the target analyte or target biomarker. Similarly, the term "label specific binding partner" will be understood to refer to any molecule capable of specifically associating with a particular label, and "biotin specific binding partner" will be understood to refer to any molecule capable of specifically associating with biotin. For example, but not by way of limitation, the means for binding / binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte / label / biotin, etc.

[0054] The term "immunoassay" as utilized herein refers to an assay to determine the presence of a diagnostic biomarker in a biological sample by reacting the sample with at least one antibody (or antigen-binding fragment thereof) that specifically binds to the diagnostic biomarker, wherein the reaction is carried out for a time and under conditions that allow for the formation of an immunocomplex between the at least one antibody (or antigen-binding fragment thereof) and the diagnostic biomarker. In certain non-limiting embodiments, thequantitative determination of such an immunocomplex is then performed. In certain particular (but non-limiting) embodiments, the immunoassays may detect an immobilized complex between a serum marker and a serum marker-binding antibody using a second antibody that is labeled and binds to the serum marker at a second epitope that does not overlap with the epitope to which the first antibody binds, so that both antibodies can bind to the same serum marker molecule, thereby forming a sandwich. In the sandwich immunoassay procedures, a serum marker-binding antibody can be a capture antibody attached to an insoluble material and the second antibody can be a labeling antibody. The above-described sandwich immunoassay procedures can be used with the antibodies described hereinafter.

[0055] Turning now to particular non-limiting embodiments of the present disclosure, embodiments of the present disclosure include individual biomarkers for pTau (i.e., a Tau biomarker wherein the Tau is phosphorylated at a specific threonine residue thereof) as well as compositions / kits / systems / devices / assays including reagents for measuring such biomarkers, along with methods of using same. In certain non-limiting embodiments, the biomarker is pTau217.

[0056] In certain non-limiting embodiments, the present disclosure includes compositions, reagents, kits, systems, and methods for determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual. The method includes performing a sandwich immunoassay with two means for binding to Tau / specific binding partners (referred to hereinafter as "antibodies," by way of example, but not by way of limitation): a capture antibody that specifically binds to an epitope containing the phosphorylated threonine residue (and thus is capable of distinguishing between phosphorylated and nonphosphorylated Tau) and a detection antibody that binds to a second epitope of Tau that is substantially non-overlapping with the epitope to which the capture antibody binds so that a sandwich immunocomplex can be formed. The capture antibody is modified so as to bind to a solid support and thus be captured thereon, whereas the detection antibody is conjugated to a detectable label for detecting the sandwich immunocomplex attached to the solid support.

[0057] In certain non-limiting embodiments, the present disclosure relates to a non-transitory computer readable medium containing executable instructions that, whenexecuted causes a processor to perform operations comprising one or more of any of the embodiments described herein.

[0058] Also provided by the present disclosure are kits including reagents containing the capture and detection antibodies described herein above or otherwise contemplated herein, along with a solid support that is modified so that the capture antibodies can bind thereto, as well as instructions for use (e.g., treatment, prophylactic, or diagnostic use). In some non-limiting embodiments, the kit is used for an in vitro diagnostic assay to evaluate, screen, detect, diagnose, and / or provide a risk prediction for Alzheimer's disease or preclinical Alzheimer's disease (and in particular (but non-limiting) embodiments, distinguish AD from other neuropathies (such as, but not limited to, ALS). In some non-limiting embodiments, the kit further includes an agent for detecting the detectable label conjugated to the detection antibody.

[0059] in some non-limiting embodiments, the kit further includes one or more control samples. In some non-limiting embodiments, the control samples include one or more Tau Biomarker standards (such as, but not limited to, pTau217).

[0060] In addition to the above, a kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and / or an agent for treating a condition or disorder described herein. Alternatively, other ingredients can be included in a kit, but in different compositions or containers than the anti-Tau Biomarker agents. In such embodiments, a kit can include instructions for admixing the anti-Tau Biomarker agents and the other ingredients, or for using the anti-Tau Biomarker together with the other ingredients.

[0061] In certain non-limiting embodiments, kits for use in accordance with the present disclosure may include one or more other components, such as (but not limited to) a reference or control sample(s), instructions for processing samples, performing tests on samples, instructions for interpreting the results, buffers, and / or other reagents necessary for performing tests.

[0062] Also provided herein are compositions. In some non-limiting embodiments, a composition includes two or more of any of the anti-Tau antibodies as described or otherwise contemplated herein (one of which specifically binds to pTau217). In certain non-limiting embodiments, the compositions include compositions shown in FIG. 1 individually, or collectively as a complex.

[0063] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing an immunoassay to evaluate, screen, detect, diagnose, and / or provide a risk prediction for Alzheimer's disease or preclin ical Alzheimer's disease; in certain particular (but non-limiting) embodiments, the kit is further capable of distinguishing AD from other neuropathic diseases and disorders (such as, but not limited to, ALS). The kit includes a first reagent (i.e., detection reagent) comprising a first label conjugated to a first anti-Tau monoclonal antibody or antigen-binding fragment thereof; a second reagent comprising a second label conjugated to a second anti-Tau monoclonal antibody or antigen-binding fragment thereof, wherein the second anti-Tau monoclonal antibody / fragment specifically binds to pTau (and in particular, specifically binds to an epitope containing the phosphorylated threonine residue and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau); and a third reagent, comprising a solid support coated with a component that specifically binds to the second label. The first and second anti-Tau monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of pTau so as to form a sandwich complex, and the component of the third reagent specifically binds to the second label of the second reagent to attach the sandwich complex to the solid support.

[0064] Certain non-limiting embodiments of the present disclosure are directed to a system for performing an immunoassay for pTau (such as, but not limited to, pTau217) wherein the system includes any of the kits disclosed or otherwise contemplated herein. The system may further include one or more other components; for example, a sample collection tube (such as, but not limited to, a serum separator tube) and / or at least one additional reagent for initiating detection of the first label.

[0065] Certain non-limiting embodiments of the present disclosure are directed to a method of determining the presence and / or concentration of pTau (such as, but not limited to, pTau217) in a biological sample. The method includes one or more of the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(d)to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to a first anti-Tau monoclonal antibody or antigen-binding fragment thereof, wherein the first monoclonal antibody or antigen-binding fragment thereof serves as a detection antibody; (c) a second reagent, comprising a second label conjugated to a second anti-Tau monoclonal antibody or antigen-binding fragment thereof, wherein the second anti-Tau monoclonal antibody orantigen-binding fragment thereof specifically binds to an epitope containing the phosphorylated threonine residue and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau, and wherein the second monoclonal antibody or antigen-binding fragment thereof serves as a capture antibody, and wherein the first and second anti- Tau monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of Tau so as to form a sandwich complex; and (d) a third reagent comprising a solid support coated with a binding partner for the second label; (2) incubating the mixture under conditions whereby (b) and (c) bind to pTau present in the biological sample to form a first sandwich complex, and the binding partner of (d) binds to the second label to attach the sandwich complex to the solid support of (d); (3) detecting a signal generated by the first label indirectly bound to the solid support of (d); and (4) determining the presence and / or concentration of pTau present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the first label indirectly bound to the solid support.

[0066] In a particular (but non-limiting) embodiment, the method is further defined as a method of determining the presence of, severity of, risk predictor for, and / or predisposition to Alzheimer's disease. In a particular (but non-limiting) embodiment, the method is further capable of distinguishing AD from other neuropathic diseases or disorders (such as, but not limited to, ALS).

[0067] Any biological fluid sample known in the art or otherwise contemplated herein that may contain pTau217 that is detectable by two or more anti-Tau antibodies as described herein and that is indicative of Alzheimer's disease may be utilized in accordance with the present disclosure. Examples of biological fluid samples that may be utilized include, but are not limited to, blood, serum, plasma, saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, combinations thereof, and the like. In certain non-limiting embodiments, the biological fluid may be altered, and / or may include human serum and plasma (EDTA and lithium heparin).

[0068] Any anti-Tau / anti-pTau antibodies known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Anti-Tau / anti-pTau antibodies are well known in the art and commercially available. For example (but not by way of limitation), anti-Tau / anti-pTau antibodies that may be utilized in accordance with the present disclosure as the antibodies bound to the first and second labels include thosedisclosed in International Patent Application Publication No. WO 2023 / 039107; and US Patent Application Publication No. 2024 / 0353428. Also, various anti-Tau / anti-pTau antibodies (as well as the commercial sources thereof) are listed in Tables 1-4 in the Examples section. In addition, anti-Tau / anti-pTau monoclonal antibodies utilizable in accordance with the present disclosure are available from various commercial sources including (but not limited to) ALZpath (Carlsbad, CA); (Fujirebio (Tokyo, Japan); ADx Neurosciences (Gent, Belgium); Bioventix (Farnham, UK; see, for example (but not by way of limitation) ADx204 (which binds to the N-terminal amino acids 6-18 of Tau)); Biolegend (San Diego, CA); Abeam (Cambridge, UK); and many others. However, this list is not inclusive, and there are many additional commercial sources of antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of monoclonal antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the antibodies or the characteristics thereof is deemed necessary.

[0069] However, the various antibodies discussed herein above is not an exclusive list of antibodies that can be utilized in accordance with the present disclosure; indeed, there are many additional commercial sources of anti-Tau / anti-pTau antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of anti-Tau / anti-pTau antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-Tau / anti-pTau antibodies or the characteristics thereof is deemed necessary.

[0070] In certain non-limiting embodiments, a suitable pTau biomarker for use herein (and to which a capture antibody utilized in accordance with the present disclosure can specifically bind) includes a polypeptide comprising the amino acid sequence of SEQ ID NO:11, wherein the threonine at position 217 thereof is phosphorylated.

[0071] As used herein, the term "sequence identity" refers to the percent identity of bases or amino acids determined by comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms having various weighting parameters. Sequence identity between two polypeptides or two polynucleotides can be determined using sequence alignment by various methods and computer programs (e.g., BLAST, FAST, L-ALIGN, etc.), available through the worldwide web at sites including GENBANK (NationalCenter for Biotechnology Information at the National Institutes of Health, Bethesda, MD) and EMBL-EBI (European Bioinformatics Institute, Wellcome Genome Campus, Hinxston, Cambridge, UK). Sequence identity between two polynucleotides or two polypeptide sequences is generally calculated using the standard default parameters of the various methods or computer programs.

[0072] Any of the antibodies or antigen-binding fragments described or otherwise contemplated herein may be labeled or otherwise conjugated to various chemical or biomolecule moieties for use in the diagnostic applications disclosed herein. The moieties may be detectable labels, for example (but not by way of limitation), chemiluminescent labels (e.g., acridinium esters and sulfonamides, luminol and isoluminol), phosphorescent labels, fluorescent labels (e.g., FITC), electrochemiluminescent labels (e.g., ruthenium (II) chelates), cloned enzyme donors, photosensitizer particles or chemiluminescer particles for luminescent oxygen channeling immunoassays (LOCI), lanthanide chelates for time-resolved fluorescence immunoassays (TR-FIA), radiolabels, biotin, digoxigenin, enzymes and the like, for example, radionuclides, such as, but not limited to, tritium, carbon-14, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111, and fissionable nuclides such as boron-10 or an actinide, and any other detectable labels / detection agents disclosed or otherwise contemplated herein. In some non-limiting embodiments, enzymes may be conjugated to the described antibodies for the purposes of detecting bound antibody in a sample. Such enzyme conjugates include, but are not limited to, alkaline phosphatase (AP), horseradish peroxidase, beta-galactosidase and glucose-6-phosphate dehydrogenase (G6PDH). Other enzymes used to determine antibody binding in solution-based immunoassays would be understood by those skilled in the art to be suitable for use as a conjugate for the antibodies described herein. In addition, compounds such as acridinium esters may also be conjugated to the provided antibodies to allow for detection in an immunoassay.

[0073] In certain particular (but non-limiting) embodiments, the first label utilized in the first reagents of the kits, systems, and methods described or otherwise contemplated herein is a detectable label. For example (but not by way of limitation), the first label can be a chemiluminescent compound (e.g., an acridinium ester compound), a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and / or an enzyme. These exemplary labels can usually only be detected when excited by methods that include, but are not limitedto, addition of different chemicals, stimulation by light, or exposure to substrate or other compounds. When using an acridinium ester compound, chemiluminescence is triggered by peroxide and acid / base, resulting in a flash that can be read by appropriate instrumentation. In addition, an optional wash step may be used before initiating detectability of the detectable label.

[0074] In a particular (but non-limiting) embodiment, the first label is an acridinium ester. In this instance, the signal generation and detection step of any of the methods disclosed or otherwise contemplated herein may comprise adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0075] The second label utilized in the second reagents of the kits, systems, and methods described or otherwise contemplated herein may be any molecule for which a specific binding partner is available that can be conjugated to the solid support, so long as the second label and its binding partner do not otherwise interfere with the assay being performed. Nonlimiting examples of second labels and specific binding partner combinations that can be utilized in the kits, systems, and methods in accordance with the present disclosure include biotin for binding to avidin, streptavidin, traptavidin, or anti-biotin antibody; fluorescein (e.g., fluorescein isothiocyanate (FITC)) for binding to anti-fluorescein antibody or fragment thereof; digoxigenin for binding to anti-digoxigenin antibody; other hapten and binding partner combinations; and the like. These types of label and specific binding partner combinations are well known in the art and widely available commercially. Thus, no further description thereof is deemed necessary.

[0076] Any solid supports known in the art and capable of functioning in accordance with the kits, systems, and methods described herein may be utilized in accordance with the present disclosure. Non-limiting examples of solid phase supports that may be utilized include magnetic particles, such as (but not limited to) magnetic latex particles; paramagnetic particles (PMPs); cross-linked dextran available under the trademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, NJ.); agarose; polystyrene beads; polyvinyl chloride; polystyrene; cross-linked polyacrylamide; nitrocellulose- or nylon-based webs such as sheets, strips, or paddles; or tubes, plates, or wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. When using magnetic or paramagnetic particles, some source of a magnetic field may be used to retain the particles and molecules bound directly or indirectlyto the particles during an optional wash step. The molecules may be bound covalently, by salt-bridges, hydrogen bonding, or another type of bond.

[0077] The compositions / reagents of the kits or systems may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and / or single aliquot form within the kit or system. Alternatively, in a particular (but nonlimiting) embodiment, one or more of the reagents may be disposed in the kit or system in the form of a single aliquot lyophilized reagent. The use of dried reagents in kits / microfluidics devices is described in detail in US Patent No. 9,244,085.

[0078] Also, the compositions / reagents present in the kits or systems may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. In addition, the kit or system may include a device (such as, but not limited to, a microfluidics device or cartridge for loading onboard of a clinical instrument) in which one or more of the compositions / reagents are disposed.

[0079] In a particular (but non-limiting) embodiment, the reagent comprising the anti- pTau antibody conjugated to the second label and the reagent comprising the solid support coated with the binding partner for the second label are combined into a single component / compartment within the kit.

[0080] The relative amounts of the various compositions / reagents in the kits or systems can vary widely to provide for concentrations of the compositions / reagents that substantially optimize the reactions that need to occur during the methods and further to optimize substantially the sensitivity and selectivity of an assay. Under appropriate circumstances, one or more of the compositions / reagents in the kit or system can be provided as a dry powder, such as a lyophilized powder, and the kit or system may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these compositions. Quality control and / or calibration reagent(s) may also be included with the kit or system. In addition, the kit or system can further include a set of written instructions (or access to electronic instructions) explaining how to use the kit or system. A kit or system of this nature can be used in any of the methods described or otherwise contemplated herein.

[0081] In certain non-limiting embodiments, the present disclosure facilitates point of care or remote evaluations, screenings, detections, diagnoses, and / or risk predictions of Alzheimer's disease and assists health care providers in monitoring the status or progress of Alzheimer's disease (and / or treatment, thereof) at. two or more time points. Significantly, the present disclosure provides health care decision makers with an alternative to potentially inaccurate assays.

[0082] In certain non-limiting embodiments, the present disclosure employs computer-implementable algorithmic methods which utilize one or more pTau-related marker values. The predictive value of the present disclosure is validated in clinical studies to monitor the status or progress of Alzheimer's disease.

[0083] In certain non-limiting embodiments, analytical methodology is applied to information obtained from a pTau sandwich immunoassay in accordance with the present disclosure and may include statistical techniques including discriminant function analysis and nonparametric regression analysis, as well as techniques such as classification trees or neural networks.

[0084] In another non-limiting embodiment, the present disclosure provides a data structure stored in a computer-readable medium that may be read by a microprocessor and that includes at least one code that uniquely identifies a linear or nonlinear function algorithm derived in a manner described herein.

[0085] In another non-limiting embodiment, the present disclosure provides a diagnostic kit including: (a) a data structure stored in a computer-readable medium that may be read by a microprocessor and that includes at least one code that uniquely identifies a linear or nonlinear function algorithm derived in a manner described herein; and (b) one or more immunoassays that detect and determine patient pTau serum marker values.

[0086] Methods and kits provided herein are able to detect pTau Biomarker in a sample with a sensitivity and a specificity that renders the outcome of the test reliable enough to be medically actionable. Methods and kits described herein for evaluation, screening, detection, diagnosis, and / or risk prediction of Alzheimer's disease in a subject detects pTau (such as, but not limited to, pTau217) with a sensitivity greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100%. In some non-limiting embodiments, methods and kits provided herein can detect the pTau biomarker (such as, but not limited to, pTau217) with asensitivity that is between about 70%-100%, between about 80%-100%, or between about 90-100%. In some non-limiting embodiments, methods and kits provided herein can detect the pTau biomarker (such as, but not limited to, pTau217) with a sensitivity and a specificity that is between about 50%-100%, between about 60%-100%, between about 70%-100%, between about 80%-100%, or between about 90-100%.

[0087] Any of the methods described or otherwise contemplated herein can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some non-limiting embodiments, a computer system may be arranged to output a pTau Biomarker (such as, but not limited to, pTau217 Biomarker) score based on receiving a pTau Biomarker profile and / or a level of one or more of any of the pTau Biomarkers disclosed or otherwise contemplated herein. Particularly, a computer program may include instructions for the system to select appropriate next steps, including additional medication, a treatment, and / or additional testing for a subject.

[0088] In some non-limiting embodiments, the computer program may be configured such that the computer system can identify a subject for further testing (e.g., Alzheimer's disease tests), identify a subject as being at risk of or having Alzheimer's disease, and / or identify a subject to receive medication based on received data (e.g., a pTau217 Biomarker profile) and use the data to calculate a pTau217 Biomarker score. A system may be able to rank-order identified next steps based on a pTau217 Biomarker profile with demographic factors and / or imaging-based biomarkers. A system may be able to adjust the rank ordering based on, e.g., a clinical response of a subject or of a family member of the subject who has or is suspected of having Alzheimer's disease.

[0089] FIG. 2 is a block diagram of one non-limiting embodiment of a computer system 1100 that can be used in the operations described above. The system 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Each of the components 1110, 1120, 1130, and 1140 are interconnected using a system bus 1150. The system may include analyzing equipment 1160 for determining a level of one or more biomarkers of the present disclosure in a sample.

[0090] In certain non-limiting embodiments, the processor 1110 is capable of processing instructions for execution within the system 1100. In one non-limiting embodiment, the processor 1110 is a single-threaded processor. In another non-limiting embodiment, the processor 1110 is a multi-threaded processor. The processor 1110 is capable of processinginstructions stored in the memory 1120 or on the storage device 1130, including for receiving or sending information through the input / output device 1140.

[0091] In certain non-limiting embodiments, the memory 1120 stores information within the system 1100. In one non-limiting embodiment, the memory 1120 is a computer-readable medium. In one non-limiting embodiment, the memory 1120 is a volatile memory unit. In another non-limiting embodiment, the memory 1120 is a non-volatile memory unit.

[0092] The storage device 1130 is capable of providing mass storage for the system 1100. In one non-limiting embodiment, the storage device 1130 is a computer-readable medium.

[0093] The input / output device 1140 provides input / output operations for the system 1100. In one non-limiting embodiment, the input / output device 1140 includes a keyboard and / or pointing device. In one non-limiting embodiment, the input / output device 1140 includes a display unit for displaying graphical user interfaces.

[0094] Additionally, certain non-limiting embodiments of the present disclosure are directed to non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including any of the methods disclosed or otherwise contemplated herein. In certain non-limiting embodiments, a non- transitory computer readable medium includes a hard drive, external hard drive, discs, CDs, DVDs, and the like that stores data. In certain non-limiting embodiments, software disposed within a physical medium is suitable for use herein.

[0095] In some non-limiting embodiments, a non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method of determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual is disclosed utilizing one or more of any of the method steps disclosed or otherwise contemplated herein.EXAMPLES

[0096] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.Example 1

[0097] One non-limiting embodiment of the immunoassay format for the detection of pTau 217 is shown in FIG. 1. pTau217, or phosphorylated Tau protein at threonine 217, is shown herein to serve as a blood biomarker that can indicate the presence of, severity of, and / or predisposition to Alzheimer's Disease (AD) and can also serve to distinguish AD from other neuropathic diseases and disorders.

[0098] The system illustrated in FIG. 1 utilizes a capture antibody that specifically binds to pTau217 (and thus is capable of distinguishing phosphorylated pTau217 from non¬ phosphorylated Tau), and the capture antibody is biotinylated with NHS-LC-Biotin. The system also utilizes magnetic particles that are coated with streptavidin for subsequent attachment of the capture antibody to the magnetic particles during the assay. The system further utilizes a detection antibody that specifically binds to Tau at an epitope that does not substantially overlap with the epitope to which the capture antibody binds (so that both antibodies can bind to the same pTau molecule), and the detection antibody is labeled with an acridinium ester (TSP-AE). When pTau217 is present in a biological sample, the two antibodies bind thereto to form a sandwich complex, and the sandwich is captured on the magnetic particles.

[0099] In one non-limiting example, the immunoassay of FIG. 1 is automatically performed by an analyzer (such as, but not limited to, the Atellica® IM analyzer (Siemens Healthineers USA, Malvern, PA)) as follows: (1) 100 μL of sample is dispensed into a cuvette; (2) 100 pl of Lite Reagent (containing the AE-labeled detection antibody) is dispensed into the cuvette, and then the cuvette is incubated for 12 minutes at 37°C; (3) 50 pl of Solid Phase (containing the streptavidin-coated magnetic particles and the biotinylated capture antibody) is dispensed into the cuvette, and then the cuvette is incubated for 5 minutes at 37°C; (4) a wash sequence is performed using Atellica® IM Wash; (5) 300 pL each of Atellica® IM Acid Reagent and Atellica® IM Base Reagent are dispensed to initiate the chemiluminescent reaction; and (6) results are reported.Example 2

[0100] Various materials that have been tested in accordance with the immunoassays and methods of the present disclosure are listed below.

[0101] Various non-limiting examples of monoclonal antibodies that can be utilized as the capture and / or detection antibodies in accordance with the immunoassays and methods of the present disclosure include those shown in Table 1. in addition, commercial sources ofeach antibody as well as various conjugations that have been tested for labeling each antibody are also listed in Table 1.TABLE 1Ab Name Target Vendor Conjugation(s) More Specific Conjugation(s)RD-085 p-Tau217 Fujirebio / Adx AE, Biotin 5x NHS-LC-LC-Biotin, 10X TSPAE-MCC 6F8 p-Tau217 Bioventix Biotin10H6 p-Tau217 Bioventix BiotinHL10 p-Tau217 ALZpath Biotin 5X NHS-LC-LC-BiotinTauJ.8B9 bd-Tau Bioventix AE, BiotinTauJ.5H3 bd-Tau Bioventix AE, BiotinTaul2 t-tau Biolegend AE 10X TSPAE-BAA10X TSPAE-MCC, 5X NHS-LC-LC-Biotin, ADx204 t-tau Fujirebio / Adx AE 10X TSPAE-BAA, 10X ADO-AE135, 10X ADO-AE155BT2 t-tau Fujirebio / Adx AE 10X TSPAE-MCCHT7 t-tau Fujirebio / Adx AE 10X TSPAE-MCCab291104 p-Tau217 Abeam AE TSPAE-MCCab274435 t-tau Abeam AE TSPAE-MCCab259552 t-tau Abeam Biotin NHS-LC-LC-Biotinab314558 p-Tau217 Abeam Biotin NHS-LC-LC-Biotinab314559 t-tau Abeam AE TSPAE-MCCab288167 p-Tau217 Abeam Biotin

[0102] One non-limiting example of a biotin that can be utilized in accordance with the immunoassays and methods of the present disclosure to label the capture antibody for subsequent attachment to the streptavidin-coated magnetic particles is NHS-LC-Biotin (succinimidyl-6-(biotinamido)hexanoate; CAS No: 9889-52-1). The structure of NHS-LC-Biotin is shown in FIG. 3.

[0103] Non-limiting examples of two acridinium esters that can be utilized to label the detection antibody in accordance with the immunoassays and methods of the present disclosure are shown in FIG. 4. These include TSPAE-TEG-MCC (left structure; Chemical formula C51H63N3O19S3; Molecular weight 1118.25) and TSPA-NHS (right structure; Chemical formula C36H38N2O17S3; Molecular weight 866.88; 21,000 A / (Mx cm) @ 410 nm @ pH2).

[0104] Various non-limiting examples of specific capture antibodies that can be utilized in accordance with the immunoassays and methods of the present disclosure are shown in Table2. All of these antibodies are hybridoma based. The binding sequence for the target analyte "pTau217-tau" is " SLP{pTHR}PP" (SEQ ID NO:14), and the binding sequence for the target analyte " Brain Tau" is " DEAAGHVTQARMVSKS" (SEQ ID NO:13).TABLE 2TargetAnalyte Antibody Specificity Isotype Commercial Source pTau217-tau 10H6 Phosphor-Thr217 Sheep IgG BioventixpTau217-tau 10C7 phospho-Thr217 Sheep IgG BioventixpTau217-tau RD-085 phospho-Thr217 mouse IgGl ADx Neurosciences pTau217-tau HL10 phospho-Thr217 mouse IgGl AlzpathBrain Tau 8B9 CNS Tau Sheep IgG BioventixBrain Tau 5H3 CNS Tau Sheep IgG Bioventix

[0105] Then in one specific (but non-limiting) example, the capture antibodies of Table 2 were conjugated to NHS-LC-Biotin of FIG. 3 using an NHS-LC- Biotin protocol in a molar excess of 5X.

[0106] Various non-limiting examples of specific detection antibodies that can be utilized in accordance with the immunoassays and methods of the present disclosure are shown in Table 3. The antibodies listed in Table 3 are hybridoma-based with the exception of RD-073, which is recombinant (whole mAb ADx204).TABLE 3Target Commercial Antibody Specificity Binding Sequence IsotypeAnalyte SourceTauN-terminal AA ADxTau ADX204 " QEFEVMEDHAGTY" IgGl6-8 Neurosciences (SEQ ID NO:12)ADxTau RD-073 N-terminal TBD IgGlNeurosciences TauSheepBrain Tau 8B9 CNS Tau " DEAAGHVTQARMVSKS" Bioventix IgG(SEQ ID NO:13)TauSheepBrain Tau 5H3 CNS Tau " DEAAGHVTQARMVSKS" BioventixIgG(SEQ ID NO:13)

[0107] Table 4 lists the various sequences of the ALZpath HL10 antibody that specifically recognizes pTau217 (sequences taken from WO 2023 / 039107, where the HL10 antibody is referred to as " Antibody 2;" in particular, SEQ. ID NOS:1-10 of this application correspond to SEQ. ID NOS:2, 7, 11, 15, 21, 25, 31, 36, 43, and 44, respectively, of WO 2023 / 039107).TABLE 4: HL10 Antibody SequencesSEQ. ID NO: Name / AA SequenceHeavy Chain CDR1 (HCDR1):1SYAMIHeavy Chain CDR2 (HCDR2):?FISRSGITYYASWAKGHeavy Chain CDR3 (HCDR3):3EFGAVGSDYYRDAFNLLight Chain CDR1 (LCDR1):4QASESINSWLSLight Chain CDR2 (LCDR2):5RASTLASLight Chain CDR3 (LCDR3):6QSYYEEDGIGYAHeavy Chain (HC) Variable Region:METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC TVSGFSLSSY7AMIWVRQAPG KGLEWIGFIS RSGITYYASW AKGRFTISKT STTVDLKMTS LTTEDTATYF CAREFGAVGS DYYRDAFNLW GPGTLVTVSSLight Chain (LC) Variable Region:MDMRAPTQLL GLLLLWLPGA RCADIVMTQT PASVEAAVGG TVTINCQASE8SINSWLSWYQQKPGQPPNLL IYRASTLASG VPSRFSGGGS GTEYTLTISD LECADAVTYY CQSYYEEDGI GYAFGGGTEV VVEHeavy Chain:METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC TVSGFSLSSY AMIWVRQAPG KGLEWIGFIS RSGITYYASW AKGRFTISKT STTVDLKMTS LTTEDTATYF CAREFGAVGS DYYRDAFNLW GPGTLVTVSS GQPKAPSVFP LAPCCGDTPS STVTLGCLVK GYLPEPVTVT WNSGTLTNGV RTFPSVRQSS9 GLYSLSSVVS VTSSSQPVTC NVAHPATNTK VDKTVAPSTC SKPTCPPPEL LGRSSVFIFP PKPKDTLMIS RTPEVTCVVV DVSQDDPEVQ FTWYINNEQV RTARP PLREQ QFNSTIRVVS TLPIAHQDWL RGKEFKCKVH NKALPAPIEK TISKARGQPL EPKVYTMGPP REELSSRSVS LTCMINGFYP SDISVEWEKN GKAEDNYKTT PAVLDSDGSY FLYSKLSVPT SEWQRGDVFT CSVMHEALHN HYTQKSISRS PGKLight Chain:MDMRAPTQLL GLLLLWLPGA RCADIVMTQT PASVEAAVGG TVTINCQASE SINSWLSWYQ QKPGQPPNLL IYRASTLASG VPSRFSGGGS GTEYTLTISD10LECADAVFYY CQSYYEEDGI GYAFGGGTEV VVEGDPVAPT VLIFPPAADQ VATGTVTIVC VANKYFPDVT VTWEVDGTTQTTGIENSKTP QNSADCTYNLSSTLTLTSTQ YNSH KEYTCK VTQGTTSVVQ SFN RG DC

[0108] In one non-limiting example of labeling the detection antibodies with TSPAE, the Bioventix SMA TauJ.5H3 / 091221 antibody was desalted over a NAP-25 column into 0.1M NaPO4-150mM NaCl-0.1% Tween-20 pH 8 (PBST8). NHS-TSPAE was dissolved @ 2mg / ml in DMSO, and a 10X MXS was added reacting @ room temperature (i.e., 21°C) for 1 hour. 10K concentrated to 1.19ml and NAP-25 purified into 2.5ml PBST8 a BCA. 10% BSA-2% NaN3 was spiked in @ l / 20th volume. An RLU chromatogram of the labeled TauJ.5H3 detection antibody was produced, as shown in FIG. 5. Then fractions 10-15 were collected and stored in an appropriate buffer such as PBS.Example 3

[0109] In this Example, a curve response was determined using HL10 pTau217 antibody labeled with NHS-LC-Biotin (Tables 1-2) as the capture antibody and TSPAE-labeled ADx204 (Tables 1 and 3) as the detection antibody. A synthetic phosphorylated Tau peptide was used as the sample. The results are shown in Table 5 and FIGS. 6-7.TABLE 5Name Signal (RLU) Concentration (pg)TauOl 1576 0Tau02 2968 0.156Tau03 3528 0.207Tau04 6207 0.502Tau05 9899 0.974Tau06 31386 4.379Tau 07 60442 10.254

[0110] FIG. 6 demonstrates the signal in function of pTau217 concentrations on the Atellica® IM analyzer.

[0111] To demonstrate the clinical performance of the pTau217 assay of this Example, forty-five confirmed Alzheimer's disease patients were evaluated along with other dementia patients, those with moderate cognitive impairment (MCI), and healthy controls (HC) using the Atellica® pTau217 assay. The outcomes were compared with those from a commercially available pTau217 assay, as shown in FIG. 7. This Figure demonstrates that the Atellica®pTau217 assay shows excellent clinical performance in separating and distinguishing AD patients from non-AD and HC patients.Example 4

[0112] This Example includes results obtained with other capture / detection antibody combinations utilized in the pTau217 immunoassay format of FIG. 1.

[0113] Table 6 and FIG. 8 provide the curve response results obtained with biotinylated RD-085 pTau 217 as capture antibody and AE-labeled ADx204 as detection antibody, with synthetic phosphorylated Tau peptide as the sample.TABLE 6Sample Target Dose RLU Result SOI 0 2789.3333 0.009 S02 5 43976 5.2 S03 10 77075.6667 9.526 S04 20 15579.333 20.009 S05 50 383241 51.161 S06 100 723567.333 99.195 S07 500 3324505 502.037508 1000 6109614.33 997.721

[0114] Table 7 provides the curve response results obtained with biotinylated Brain derived TauJ.8B9 or TauJ.5H3 as capture antibody and AE-labeled ADx204 as detection antibody.TABLE 78B9 / ADX204 5H3 / ADX204Sample RLU Sample RLU0 GSK01 7705 GSK01 13727BD-Tau 0.1 GSK02 10680 GSK02 20311Assays 0.5 GSK03 21570 GSK03 409841 GSK04 35213 GSK04 694555 GSK05 144967 GSK05 28058510 GSK06 290442 GSK06 571056

[0115] Table 8 and FIG. 9 provide the curve response results obtained with biotinylated 10H6 pTau217 as capture antibody and AE-labeled ADx204 or TauJ.5H3 as detection antibody, with synthetic phosphorylated Tau peptide GSK3beta as the sample.TABLE 810H6 / ADx204 10H6 / 5H3Sample RLU Sample RLU0 GSK01 3719 GSK01 5114BD-Tau 0.1 GSK02 3836 GSK02 5167Assays 0.5 GSK03 5093 GSK03 63491 GSK04 6422 GSK04 78675 GSK05 18267 GSK05 1982710 GSK06 34186 GSK06 35554Example 5

[0116] p-Tau217, in both cerebrospinal fluid and plasma, is a promising diagnostic biomarker that closely correlates with the hallmark pathology of Alzheimer's disease (AD). Development of an AD specific blood-based immunoassay on a high-throughput, automated central laboratory platform offers a scalable solution for screening, diagnosis, and monitoring of AD. This Example evaluated automated p-Tau217, brain-derived Tau (bd-Tau), and total Tau (t-Tau) assays, with a focus on their specificity for detecting AD in plasma samples in the context of other neuropathies with high tau levels.

[0117] Five assay formats were compared: Advantage PLUS (Quanterix Corporation, Billerica, MA) on the Quanterix Simoa instrument utilizing a pTau217 specific antibody from ALZpath (Carlsbad, CA), and four new assays developed for the Siemens Atellica® immunoassay module (targeting p-Tau217, bd-Tau, and tTau). These various Tau proteoforms were measured in human plasma from patients with cognitive impairment or early dementia due to Alzheimer’s disease (AD, n=25), mild cognitive impairment or early dementia without Alzheimer's disease (MCI non-AD n=31), amyotrophic lateral sclerosis (ALS, n=10), and healthy controls (HC, n=13).

[0118] The data showed that all assays effectively differentiated MCl-non-AD and HC from AD. However, the newly developed assays demonstrated an advantage over existingassays due to their ability to differentiate AD from ALS. Variability in false positive rates for the different assays was also observed.

[0119] These results indicate that the disclosed immunoassays targets tauopathy in AD with greater specificity than existing methods.Example 6

[0120] This Example describes the use of the plasma pTau217 assay disclosed herein to help detect amyloid pathology in people with possible cognitive decline.

[0121] In particular, this Example describes the data development and statistical analyses for establishing cut-offs for the plasma p-Tau217 assay in relation to amyloid pathology., as quantified by amyloid-PET Centiloid values. This Example supports the use of this assay as an aid to identifying amyloid pathology associated with Alzheimer's disease and is intended for use (for example, but not by way of limitation) in specialized care or clinical settings, where results are interpreted in conjunction with other clinical and biomarker information.

[0122] Certain non-limiting objectives accomplished by this Example were to derive two diagnostic cutoffs for plasma p-Tau217 assay that achieve sensitivity >90% and specificity >90% with an intermediate zone proportion not exceeding 20% of the total population in classifying amyloid-PET Centiloid-based amyloid pathology status, and to validate the diagnostic performance of the derived pTau-217 cutoffs to confirm that the thresholds achieve the predefined sensitivity and specificity targets.

[0123] Retrospective de-identified plasma samples were obtained from multiple source cohorts, including Wisconsin Registry for Alzheimer Prevention (WRAP), the Yale Alzheimer's Disease Research Center (ADRC) / Biofinder, and the University of Wisconsin ADRC. The plasma samples were a mixture of research participants who are cognitively healthy or cognitively impaired and included a total of 5,793 longitudinal plasma samples across all cohorts (from 2,147 total subjects). The inclusion criteria for analysis were that the participant was at least 50 years old and cognitively impaired based on available clinical assessments. The exclusion criteria were less than 50 years old, implausible or missing p-Tau217 values, and / or implausible or missing amyloid-PET Centiloid values. Of these 2,147 subjects, subjects with missing diagnosis, an age less than 50, or with missing Centiloid values were excluded, thereby providing an analysis population of 139. One analytic visit per participant was selected based on a number of factors.

[0124] A primary outcome is amyloid pathology status, derived from amyloid-PET Centiloid values. Participants were classified as amyloid positive or amyloid negative based on a predefined Centiloid threshold of 24.1, which represents the transition point between normal and abnormal amyloid burden. Amyloid positive is detected as a Centiloid value ≥24.1, which is consistent with individuals who are likely to have amyloid pathology, indicative of abnormal cortical amyloid burden. Amyloid negative is detected as a Centiloid value <24.1, which is consistent with individuals who are unlikely to have amyloid pathology.

[0125] The primary variable of interest was plasma pTau-217 concentration, which was measured using the assays of the present disclosure on the Atellica® analyzer (Siemens Healthineers USA, Malvern, PA). Primary analysis used log-transformed values of pTau-217 concentration. p-Tau217 values at or above the assay's limit of detection (LoD) value of 0.0222 pg / mL were retained as reported, and values below 0.0222 pg / mL were set to 0.0222. The derivation and validation of the pTau-217 assay used the aforementioned amyloid pathology status as the comparator for assessing the diagnostic performance of pTau-217.

[0126] Statistical analysis involved a descriptive summary of demographic and clinical characteristics, derivation of p-Tau217 cutoffs, validation of p-Tau217 cutoffs, and predictive value and likelihood ratio analyses. Demographic summaries were generated for: (i) the overall analysis population (overall, by amyloid status and diagnostic subgroup); and (2) the cutoff derivation and validation populations (each summarized overall and stratified by amyloid status and diagnostic subgroup). Cross-tabulation of diagnostic subgroups and amyloid pathology status by population was determined, along with the distribution of plasma pTau-217 concentration by amyloid pathology status and population. Continuous variables were summarized using mean (SD), and categorical variables were summarized using counts and percentages.

[0127] The cutoff derivation approach included univariate logistic regression model fit using plasma p-Tau217 concentration as the sole predictor (Martinez-Dubarbie et al. (2025) Alzheimer's research & therapy, 17(1):68). A two-graph Receiver Operating Characteristic (TG ROC) curve was constructed from predicted probabilities (Greiner et al., (1995) Journal of immunological methods, 185(1):123-132), and cutoffs were selected from the TG-ROC curve based on the chosen sensitivity and specificity.c1= max {xt: sensitivity at t ≥ 0.90}c2= min {xt: specificity at t ≥ 0.90}Bootstrap resampling (1000 replicates) was performed to assess and quantify variation in cutoffs.

[0128] Validation analysis was performed using the cutoff validation population, defined as the remaining 30% stratified sample from each site with preserved amyloid pathology status distribution. The finalized cutoffs (c1and c2) were applied to the independent validation subset, and each participant was classified as: Amyloid-negative (X,j < cj); Amyloid-positive (Xu > C2); or Intermediate (ci < Xu < cz}. In addition, the following standard diagnostic performance metrics were calculated: sensitivity, specificity, PPV (Positive Predictive Value), NPV (Negative Predictive Value), AUC (Area Under the Curve), accuracy, and IZP (Indeterminate Zone Proportion).

[0129] Using the fixed plasma p-Tau217 cutoffs, participants were classified into three categories: positive, intermediate, and negative. 95% confidence intervals for likelihood ratios were calculated using an asymptotic method for the ratio of two independent binomial proportions and 95% confidence intervals for predictive values were derived by applying Bayes' theorem to the lower and upper confidence limits of the corresponding likelihood ratio.

[0130] Table 9 provides a demographic summary of the overall analysis population. The cutoff derivation population is 95, whereas the cutoff validation population is 44.TABLE 9

[0131] Table 10 provides a comparison of amyloid pathology status versus diagnostic groups by population.TABLE 10

[0132] The derived cutoff's are:Rule-out cutoff (c1): 0.49Rule-in cutoff (c2): 0.57

[0133] These cutoffs define three result categories: negative, intermediate, and positive. Table 11 illustrates the variability in the pTau217 cutoffs based on 1000 bootstrap samples, and Table 12 provides performance of cutoffs in the validated population.TABLE 11Cutoffs C1C2Min. 0.4891 0.5691stQu. 0.4907 0.5703Median 0.4908 0.5703Mean 0.4908 0.57033rdQu. 0.4908 0.5703Max. 0.4909 0.5704TABLE 12Metric Estimate Lower Limit Upper Limit Sensitivity 0.9333 0.7868 0.9815 Specificity 0.9 0.5958 0.9821PPV 0.9655 0.8282 0.9939NPV 0.8182 0.523 0.9486 Accuracy 0.925 0.8014 0.9742IZP 0.0909 0.0359 0.2116 AUC 0.8352 0.9394 1

[0134] In conclusion, dual cutoff’s for pTau217 were derived and validated using the internal dataset. Positive results provide high predictive value for amyloid positivity, and negative results provide strongly reduced likelihood of amyloid pathology. Thus, the results of this Example demonstrate the diagnostic utility of pTau217 cutoffs as an aid for identifying amyloid pathology.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0135] Illustrative Embodiment 1. A kit for performing an immunoassay for a phosphorylated Tan biomarker (pTau), the kit comprising: a first reagent comprising a first means for binding to Tau, wherein the first means for binding is conjugated to a detection label; a second reagent comprising a second label conjugated to a second means for binding to Tau, wherein the second means for binding to Tau specifically binds to an epitope of Tau that contains the phosphorylated threonine residue and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau; a third reagent comprising a solid support coated with a binding partner for the second label; and wherein the first and second means for binding to Tau bind to substantially non-overlapping epitopes of Tau so as to form a sandwich complex, and the second label binding partner coated on the solid support of the third reagent binds to the second label ofthe second reagentto attach the sandwich complex to the solid support.

[0136] Illustrative Embodiment 2. The kit of Illustrative Embodiment 1, wherein the pTau is pTau217.

[0137] Illustrative Embodiment 3. The kit of Illustrative Embodiment 1 or 2, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0138] Illustrative Embodiment. 4. The kit of Illustrative Embodiment 3, wherein the first label is an acridinium ester.

[0139] Illustrative Embodiment 5. The kit of Illustrative embodiment 4, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

[0140] Illustrative Embodiment 6. The kit of any of Illustrative Embodiments 1-5, wherein the second label is selected from the group consisting of biotin, fluorescein, and digoxigenin.

[0141] Illustrative Embodiment 7. The kit of Illustrative Embodiment 6, wherein the second label is biotin, and wherein the second label binding partner coated on the solidsupport is selected from the group consisting of avidin, streptavidin, traptavidin, and an antibiotin antibody or antigen-binding fragment thereof.

[0142] illustrative Embodiment 8. The kit of illustrative Embodiment 6, wherein the second label is a fluorescein, and wherein the second label binding partner coated on the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0143] Illustrative Embodiment 9. The kit of Illustrative Embodiment 8, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0144] Illustrative Embodiment 10. The kit of Illustrative Embodiment 6, wherein the second label is digoxigenin, and wherein the second label binding partner coated on the solid support is an anti-digoxigenin antibody.

[0145] illustrative Embodiment 11. The kit of any of Illustrative Embodiments 1-10, wherein the solid support comprises magnetic particles.

[0146] illustrative Embodiment 12. The kit of any of Illustrative Embodiments 1-11, wherein the second and third reagents are combined into a single component within the kit.

[0147] Illustrative Embodiment 13. The kit of any of Illustrative Embodiments 1-12, wherein the Tau to which the first means for binding binds is further defined as the human Tau-441 isoform.

[0148] Illustrative Embodiment 14. The kit of any of Illustrative Embodiments 1-13, wherein the first means for binding and the second means for binding each binds to a portion of SEQ ID NO: 11.

[0149] Illustrative Embodiment 15. The kit of any of Illustrative Embodiments 1-14, wherein the first means for binding binds to SEQ ID NO:12 or 13.

[0150] Illustrative Embodiment 16. The kit of any of Illustrative Embodiments 1-15, wherein the second means for binding binds to SEQ ID NO:14.

[0151] Illustrative Embodiment 17. The kit of any of Illustrative Embodiments 1-16, wherein the first means for binding to Tau is a first monoclonal antibody or antigen-binding fragment thereof, and the second means for binding to Tau is a second monoclonal antibody or antigen -binding fragment thereof.

[0152] Illustrative Embodiment 18. The kit of Illustrative Embodiment 17, wherein the first monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of ADx204, RD-073, 8B9, and 5H3 or an antigen-binding fragment thereof.

[0153] Illustrative Embodiment 19. The kit of any of Illustrative Embodiments 17-18, wherein the second monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of 10H6, 10C7, RD-085, HL10, 8B9, 6F8, and 5H3, or an antigen¬ binding fragment thereof.

[0154] Illustrative Embodiment 20. The kit of any of Illustrative Embodiments 17-19, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

[0155] Illustrative Embodiment 21. The kit of any of Illustrative Embodiments 17-20, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:7 and a light chain variable region as set forth in SEQ ID NO:8.

[0156] Illustrative Embodiment 22. The kit of any of Illustrative Embodiments 17-21, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO:10.

[0157] Illustrative Embodiment 23. The kit of any of Illustrative embodiments 17-22, wherein the second monoclonal antibody or antigen -binding fragment thereof comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences from WO 2023 / 039107.

[0158] Illustrative Embodiment 24. The kit of any of Illustrative embodiments 17-23, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of light chain variable region and heavy chain variable region amino acid sequences from WO 2023 / 039107 (including amino acid sequences that are at least about 90% identical to specific sequences disclosed therein).

[0159] Illustrative Embodiment 25. A system for performing an immunoassay for pTau, the system comprising: the kit of any one of Illustrative Embodiments 1-24.

[0160] Illustrative Embodiment 26. The system of Illustrative Embodiment 25, further comprising at least one sample collection tube.

[0161] Illustrative Embodiment 27. The system of Illustrative Embodiment 26, wherein the sample collection tube is a serum separator tube.

[0162] Illustrative Embodiment 28. The system of any of Illustrative Embodiments 25-2.7, further comprising at least one additional reagent for initiating detection ofthe first label.

[0163] Illustrative Embodiment 29. The system of Illustrative Embodiment 28, wherein the first label comprises an acridinium ester, and wherein the at least one additional reagent initiates detection of chemiluminescence triggered by the acridinium ester.

[0164] Illustrative Embodiment 30. The system of Illustrative Embodiment 28 or 29, wherein the at least one additional reagent comprises an acid and a base.

[0165] Illustrative Embodiment 31. The kit of any of Illustrative Embodiments 1-24 or the system of any of Illustrative Embodiments 25-30, further defined as being capable of detecting the concentration of pTau at a sensitivity of at least about 90% and / or a specificity of at least about 90%.

[0166] Illustrative Embodiment 32. The kit / system of Illustrative Embodiment 31, wherein the sensitivity and specificity are obtained at a rule-out cutoff (ct) of 0.49 and / or a rule-in cutoff (c2) of 0.57.

[0167] Illustrative Embodiment 33. A method of determining the presence and / or concentration of pTau in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(d)to form a mixture: (a) a biological sample; (b) a first reagent comprising a first means for binding to Tau, wherein the first means for binding is conjugated to a detection label; (c) a second reagent comprising a second label conjugated to a second means for binding to Tau, wherein the second means for binding to Tau specifically binds to an epitope of Tau that contains the phosphorylated threonine residue and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau; and (d) a third reagent comprising a solid support coated with a binding partnerforthe second label; (2) incubating the mixture under conditions whereby (b) and (c) bind to pTau present in the biological sample to form a sandwich complex, and the second label binding partner of (d) binds to the second label of (c) to attach the sandwich complex to the solid support of (d); (3) detecting a signal generated by the first label indirectly bound to the solid support of (d); and (4) determining the presence and / or concentration of pTau present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the first label indirectly bound to the solid support.

[0168] Illustrative Embodiment 34. The method of Illustrative Embodiment 33, wherein the pTau is pTau217.

[0169] Illustrative Embodiment 35. The method of Illustrative Embodiment 33 or 34, wherein the biological sample is selected from the group consisting of blood, serum, plasma,saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, and combinations thereof.

[0170] Illustrative Embodiment 36. The method of any of' Illustrative Embodiments 33-35, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0171] Illustrative Embodiment 37. The method of Illustrative Embodiment 36, wherein the first label is an acridinium ester.

[0172] Illustrative Embodiment 38. The method of Illustrative embodiment 37, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

[0173] Illustrative Embodiment 39. The method of any of Illustrative Embodiments 33-38, wherein the second label is selected from the group consisting of biotin, fluorescein, and digoxigenin.

[0174] Illustrative Embodiment 40. The method of Illustrative Embodiment 39, wherein the second label is biotin, and wherein the second label binding partner coated on the solid support is selected from the group consisting of avidin, streptavidin, traptavidin, and an anti¬ biotin antibody or antigen-binding fragment thereof.

[0175] Illustrative Embodiment 41. The method of Illustrative Embodiment 39, wherein the second label is a fluorescein, and wherein the second label binding partner coated on the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0176] Illustrative Embodiment 42. The method of Illustrative Embodiment 41, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0177] Illustrative Embodiment 43. The method of Illustrative Embodiment 39, wherein the second label is digoxigenin, and wherein the second label binding partner coated on the solid support is an anti-digoxigenin antibody.

[0178] Illustrative Embodiment 44. The method of any of Illustrative Embodiments 33-43, wherein the solid support comprises magnetic particles.

[0179] Illustrative Embodiment 45. The method of any of Illustrative Embodiments 33-44, wherein the Tau to which the first means for binding binds is further defined as the human Tau-441 isoform.

[0180] Illustrative Embodiment 46. The method of any of Illustrative Embodiments 33-45, wherein the first means for binding and the second means for binding each binds to a portion of SEQ ID NO:11.

[0181] Illustrative Embodiment 47. The method of any of Illustrative Embodiments 33-46, wherein the first means for binding binds to SEQ ID NO:12 or 13.

[0182] Illustrative Embodiment 48. The method of any of Illustrative Embodiments 33-47, wherein the second means for binding binds to SEQ ID NO:14.

[0183] Illustrative Embodiment 49. The method of any of Illustrative Embodiments 33-48, wherein the first means for binding to Tau is a first monoclonal antibody or antigen-binding fragment thereof, and the second means for binding to Tau is a second monoclonal antibody or antigen-binding fragment thereof.

[0184] Illustrative Embodiment 50. The method of Illustrative Embodiment 49, wherein the first monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of ADx204, RD-073, 8B9, and 5H3 or an antigen-binding fragment thereof.

[0185] Illustrative Embodiment 51. The method of any of Illustrative Embodiments 49-50, wherein the second monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of 10H6, 10C7, RD-085, HL10, 8B9, 6F8, and 5H3, or an antigenbinding fragment thereof.

[0186] Illustrative Embodiment 52. The method of any of Illustrative Embodiments 49-51, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

[0187] Illustrative Embodiment 53. The method of any of Illustrative Embodiments 49-52, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:7 and a light chain variable region as set forth in SEQ ID NO:8.

[0188] Illustrative Embodiment 54. The method of any of Illustrative Embodiments 49-53, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO:10.

[0189] Illustrative Embodiment 55. The method of any of Illustrative embodiments 49-54, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises anycombination of light chain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences from WO 2023 / 039107.

[0190] Illustrative Embodiment 56. The method of any of Illustrative embodiments 49-55, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of light chain variable region and heavy chain variable region amino acid sequences from WO 2023 / 039107 (including amino acid sequences that are at least about 90% identical to specific sequences disclosed therein).

[0191] Illustrative Embodiment 57. The method of any of Illustrative Embodiments 33-56, further comprising the step of performing a separation of the biological sample prior to step (1) using at. least one sample collection tube.

[0192] Illustrative Embodiment 58. The method of Illustrative Embodiment 57, wherein the sample collection tube is a serum separator tube.

[0193] illustrative Embodiment 59. The method of any of Illustrative Embodiments 33-58, further comprising performing a wash step prior to step (3).

[0194] Illustrative Embodiment 60. The method of any of Illustrative Embodiments 33-59, wherein the first label is an acridinium ester, and wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0195] Illustrative Embodiment 61. The method of any of Illustrative Embodiments 33-60, further defined as a method of determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual.

[0196] illustrative Embodiment 62. The method of any of Illustrative Embodiments 33-61, wherein the concentration of pTau is detected at a sensitivity of at least about 90% and / or a specificity of at least about 90%.

[0197] Illustrative Embodiment 63. The method of Illustrative Embodiment 62, wherein the sensitivity and specificity are obtained at a rule-out cutoff (c₁) of 0.49 and / or a rule-in cutoff (c₂) of 0.57.

[0198] Illustrative Embodiment 64. A kit for performing an immunoassay for a Tau biomarker, wherein the Tau is phosphorylated at a specific threonine residue thereof (pTau), the kit comprising: a first reagent comprising a first monoclonal antibody or antigen-binding fragment thereof, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to Tau, and wherein the first monoclonal antibody or antigen-bindingfragment thereof is conjugated to an acridinium ester; a second reagent comprising a second monoclonal antibody or antigen-binding fragment thereof, wherein the second monoclonal antibody or antigen-binding fragment thereof specifically binds to an epitope containing the specific phosphorylated threonine residue ofTau (pTau) and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau, and wherein the second monoclonal antibody or antigen-binding fragment thereof is biotinylated; a third reagent comprising a solid support coated with a biotin specific binding partner; and wherein the first and second monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of Tau so as to form a sandwich complex, and the biotin specific binding partner of the third reagent binds to the biotinylated second monoclonal antibody or antigen-binding fragment thereof to attach the sandwich complex to the solid support.

[0199] Illustrative Embodiment 65. The kit of Illustrative Embodiment 64, wherein the pTau is pTau217.

[0200] Illustrative Embodiment 66. The kit of Illustrative Embodiment 64 or 65, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to human Tau-441 isoform.

[0201] Illustrative Embodiment 67. The kit of any of Illustrative Embodiments 64-66, wherein the first monoclonal antibody or antigen-binding fragment thereof binds to SEQ ID NO:12 or 13.

[0202] Illustrative Embodiment 68. The kit of any of Illustrative Embodiments 64-67, wherein the second monoclonal antibody or antigen-binding fragment thereof binds to SEQ ID NO:14.

[0203] Illustrative Embodiment 69. The kit of any of Illustrative Embodiments 64-68, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

[0204] Illustrative Embodiment 70. The kit of any of Illustrative Embodiments 64-69, wherein the biotin specific binding partner of the third reagent is selected from the group consisting of avidin, streptavidin, traptavidin, and an anti-biotin antibody or antigen-binding fragment thereof.

[0205] Illustrative Embodiment 71. The kit of any of Illustrative Embodiments 64-70, wherein the solid support comprises magnetic particles.

[0206] Illustrative Embodiment 72. The kit of any of Illustrative Embodiments 64-71, wherein the first monoclonal antibody or antigen-binding fragment thereof is selected fromthe group consisting of ADx204, RD-073, 8B9, and 5H3 or an antigen-binding fragment thereof.

[0207] Illustrative Embodiment 73. The kit of any of Illustrative Embodiments 64-72, wherein the second monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of 10H6, 10C7, RD-085, HL10, 8B9, 6F8, and 5H3, or an antigenbinding fragment thereof.

[0208] Illustrative Embodiment 74. The kit of any of Illustrative Embodiments 64-73, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

[0209] Illustrative Embodiment 75. The kit of any of Illustrative Embodiments 64-74, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:7 and a light chain variable region as set forth in SEQ ID NO:8.

[0210] Illustrative Embodiment 76. The kit of any of Illustrative Embodiments 64-75, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO:10.

[0211] Illustrative Embodiment 77. The kit of any of Illustrative embodiments 64-76, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences from WO 2023 / 039107.

[0212] Illustrative Embodiment 78. The kit of any of Illustrative embodiments 64-77, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of light chain variable region and heavy chain variable region amino acid sequences from WO 2023 / 039107 (including amino acid sequences that are at least about.90% identical to specific sequences disclosed therein).

[0213] Illustrative Embodiment 79. The kit. of any of Illustrative Embodiments 64-78, further defined as being capable of detecting the concentration of pTau at a sensitivity of at least about 90% and / or a specificity of at least about 90%.

[0214] illustrative Embodiment 80. The kit of Illustrative Embodiment 79, wherein the sensitivity and specificity are obtained at a rule-out cutoff (c₁) of 0.49 and / or a rule-in cutoff (c₂) of 0.57.

[0215] Illustrative Embodiment 81. A method of determining the presence and / or concentration of pTau in a biological sample., the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first monoclonal antibody or antigen-binding fragment thereof, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to Tau, and wherein the first monoclonal antibody or antigen¬ binding fragment thereof is conjugated to an acridinium ester; (c) a second reagent comprising a second monoclonal antibody or antigen-binding fragment thereof, wherein the second monoclonal antibody or antigen-binding fragment thereof specifically binds to an epitope containing the specific phosphorylated threonine residue of Tau (pTau) and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau, and wherein the second monoclonal antibody or antigen-binding fragment thereof is biotinylated; and (d) a third reagent comprising a solid support coated with a biotin specific binding partner; (2) incubating the mixture under conditions whereby (b) and (c) bind to pTau present in the biological sample to form a sandwich complex, and the biotin specific binding partner of (d) binds to the biotin of (c) to attach the sandwich complexes to the solid support of (d); (3) detecting a signal generated by the acridinium ester indirectly bound to the solid support of (d); and (4) determining the presence and / or concentration of pTau present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the acridinium ester indirectly bound to the solid support.

[0216] Illustrative Embodiment 82. The method of Illustrative Embodiment 81, wherein the pTau is pTau217.

[0217] Illustrative Embodiment 83. The method of Illustrative Embodiment 81 or 82, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to human Tau-441 isoform.

[0218] Illustrative Embodiment 84. The method of any of Illustrative Embodiments 81-83, wherein the first monoclonal antibody or antigen-binding fragment thereof binds to SEQ ID NO:12 or 13.

[0219] Illustrative Embodiment 85. The method of any of Illustrative Embodiments 81-84, wherein the second monoclonal antibody or antigen-binding fragment thereof binds to SEQ ID NO: 14.

[0220] Illustrative Embodiment 86. The method of any of Illustrative Embodiments 81-85, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, and combinations thereof.

[0221] Illustrative Embodiment 87. The method of any of Illustrative Embodiments 81-86, wherein step (3) comprises adding at. least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0222] Illustrative Embodiment 88. The method of any of Illustrative Embodiments 81-87, further defined as a method of determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual.

[0223] Illustrative Embodiment 89. The method of any of Illustrative Embodiments 81- 88, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

[0224] Illustrative Embodiment 90. The method of any of Illustrative Embodiments 81-89, wherein the biotin specific binding partner of the third reagent is selected from the group consisting of avidin, streptavidin, traptavidin, and an anti-biotin antibody or antigen-binding fragment thereof.

[0225] Illustrative Embodiment 91. The method of any of Illustrative Embodiments 81-90, wherein the solid support comprises magnetic particles.

[0226] Illustrative Embodiment 92. The method of any of Illustrative Embodiments 81-91, wherein the first monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of ADx204, RD-073, 8B9, and 5H3 or an antigen-binding fragment thereof.

[0227] Illustrative Embodiment 93. The method of any of Illustrative Embodiments 81-92, wherein the second monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of 10H6, 10C7, RD-085, HL10, 8B9, 6F8, and 5H3, or an antigen¬ binding fragment thereof.

[0228] Illustrative Embodiment 94. The method of any of Illustrative Embodiments 81-93, wherein the second monoclonal antibody or antigen -binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

[0229] Illustrative Embodiment 95. The method of any of Illustrative Embodiments 81-94, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises aheavy chain variable region as set forth in SEQ ID NO:7 and a light chain variable region as set forth in SEQ ID NO:8.

[0230] Illustrative Embodiment 96. The method of any of Illustrative Embodiments 81-95, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO:10.

[0231] Illustrative Embodiment 97. The method of any of Illustrative embodiments 81-96, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of light chain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences from WO 2023 / 039107.

[0232] Illustrative Embodiment 98. The method of any of Illustrative embodiments 81-97, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises any combination of light chain variable region and heavy chain variable region amino acid sequences from WO 2023 / 039107 (including amino acid sequences that are at least about 90% identical to specific sequences disclosed therein).

[0233] Illustrative Embodiment 99. The method of any of Illustrative Embodiments 81-98, further comprising performing a wash step prior to step (3).

[0234] Illustrative Embodiment 100. The method of any of Illustrative Embodiments 81- 99, wherein the concentration of pTau is detected at a sensitivity of at least about 90% and / or a specificity of at least about 90%.

[0235] Illustrative Embodiment 101, The method of Illustrative Embodiment 100, wherein the sensitivity and specificity are obtained at a rule-out cutoff (c1) of 0.49 and / or a rule-in cutoff (c2) of 0.57.

[0236] Illustrative Embodiment 102. A non-transitory computer readable medium containing executable instructions that when executed cause a processor to perform operations including the method of any one of Illustrative Embodiments 33-63 and 81-101.

[0237] Thus, in accordance with the present disclosure, there have been provided compositions, devices, and kits, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it. is intended to embraceall such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

CLAIMS1. A kit for performing an immunoassay for a Tau biomarker, wherein the Tau is phosphorylated at a specific threonine residue thereof (pTau), the kit comprising:a first reagent comprising a first monoclonal antibody or antigen-binding fragment thereof, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to Tau, and wherein the first monoclonal antibody or antigen-binding fragment thereof is conjugated to an acridinium ester; a second reagent comprising a second monoclonal antibody or antigen-binding fragment thereof, wherein the second monoclonal antibody or antigen-binding fragment thereof specifically binds to an epitope containing the specific phosphorylated threonine residue of Tau (pTau) and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau, and wherein the second monoclonal antibody or antigen-binding fragment thereof is biotinylated;a third reagent comprising a solid support coated with a biotin specific binding partner;andwherein the first and second monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of Tau so as to form a sandwich complex, and the biotin specific binding partner of the third reagent binds to the biotinylated second monoclonal antibody or antigen-binding fragment thereof to attach the sandwich complex to the solid support.

2. The kit of claim 1, wherein the pTau is pTau217.

3. The kit of claim 1, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to human Tau-441 isoform.

4. The kit of claim 1, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

5. The kit of claim 1, wherein the biotin specific binding partner of the third reagent is selected from the group consisting of avidin, streptavidin, traptavidin, and an anti-biotin antibody or antigen-binding fragment thereof.

6. The kit of claim 1, wherein the solid support comprises magnetic particles.

7. The kit of claim 1, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

8. A method of determining the presence and / or concentration of pTau in a biological sample, the method comprising the steps of:(1) combining, either simultaneously or wholly or partially sequentially, (a)-(d) to form a mixture:(a) a biological sample;(b) a first reagent comprising a first monoclonal antibody or antigenbinding fragment thereof, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to Tau, and wherein the first monoclonal antibody or antigen-binding fragment thereof is conjugated to an acridinium ester;(c) a second reagent comprising a second monoclonal antibody or antigenbinding fragment thereof, wherein the second monoclonal antibody or antigen-binding fragment thereof specifically binds to an epitope containing the specific phosphorylated threonine residue of Tau (pTau) and thus is capable of distinguishing phosphorylated Tau from unphosphorylated Tau, and wherein the second monoclonal antibody or antigen-binding fragment thereof is biotinylated; and(d) a third reagent comprising a solid support coated with a biotin specific binding partner;(2) incubating the mixture under conditions whereby (b) and (c) bind to pTau present in the biological sample to form a sandwich complex, and the biotinspecific binding partner of (d) binds to the biotin of (c) to attach the sandwich complexes to the solid support of (d );(3) detecting a signal generated by the acridinium ester indirectly bound to the solid support of (d); and(4) determining the presence and / or concentration of pTau present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the acridinium ester indirectly bound to the solid support.

9. The method of claim 8, wherein the pTau is pTau217.

10. The method of claim 8, wherein the first monoclonal antibody or antigen-binding fragment thereof specifically binds to human Tau-441 isoform.

11. The method of claim 8, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, and combinations thereof.

12. The method of claim 8, wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

13. The method of claim 8, further defined as a method of determining the presence of, severity of, and / or predisposition to Alzheimer's disease in an individual.

14. The method of claim 8, wherein the acridinium ester comprises trisulfopropyl acridinium ester (TSPAE).

15. The method of claim 8, wherein the biotin specific binding partner of the third reagent is selected from the group consisting of avidin, streptavidin, traptavidin, and an anti-biotin antibody or antigen-binding fragment thereof.

16. The method of claim 8, wherein the solid support comprises magnetic particles.

17. The method of claim 8, wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NOS:1, 2, 3, 4, 5, and 6, respectively.

18. The method of claim 8, wherein the concentration of pTau is detected at a sensitivity of at least about 90% and / or a specificity of at least about 90% at a rule-out cutoff (c1) of 0.49 and / or a rule-in cutoff (c2) of 0.57.

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