Sandwich assay for TSH biomarker detection and methods of producing and using same

Abstract

Compositions, kits, systems, and methods for determining a total TSH concentration in a biological sample and / or determining the presence, severity, and / or predisposition of thyroid disease and / or pituitary disorder in an individual are disclosed. The compositions, kits, systems, and methods utilize at least three antibodies or antigen-binding fragments thereof that specifically bind to TSH, wherein at least one of the three antibodies or antigen-binding fragments thereof specifically binds to a TSH variant so that at least antibody-TSH-antibody sandwich immunocomplexes and antibody-TSH variant-antibody sandwich immunocomplexes can be formed. The methods include determining an amount of total TSH present in the biological sample based on the amount of the two sandwich immunocomplexes formed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application ciaims benefit of US Provisional Application No. 63 / 562,449, filed March 7, 2024. The entire contents of the above-referenced patent application^) 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 EFS-Web in computer readable form as an XML file. The Sequence Listing,, created February 24, 2025, is named "202404459_Sequence_ Listing" and is 4,803 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference. BACKGROUND [GOGS] 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 of mortality. A biomarker should be easily acquired, reliably measured, and available for serial monitoring. Ideally, a biomarker would also provide an advantage of currently used clinical measures in ease, timeframe, and / or expense.

[0005] However, while tests are available for detecting biomarkers, biomarker variants have been identified that may be less easily or less accurately measured in a particular immunoassay. For example, thyroid stimulating hormone (TSH), a known biomarker, may be present in a biological sample in a mutated variant form having a mutation in the betasubunit, problematically reducing detectability in some immunoassays. For example, Shaki, et ai. (fur Thyroid J., 2022, ll(l)e210072) explains that homozygosity for the TSHB p.R75G variant abrogates TSH detection by some immune detection-based platforms, leading to a misdiagnosis of hyperthyroidism.

[0006] Therefore, there is a need in the art for new and improved immunoassays for biomarker-of-interest detection, wherein the biomarker-of-interest may include one or more isoforms, variants, or alterations which may reduce the efficacy and / or accuracy of the assay. It is to such biomarkers, as well as compositions / kits / systems / devices / assays containing reagents for measuring such biomarkers, along with methods of using same, that the present disclosure is directed. BRIEF DESCRIPTION OF THE DRAWINGS

[8087] 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 an immunoassay constructed in accordance with the present disclosure and utilizing a first capture monoclonal antibody (mAb) and a second capture mAb in a solid phase reagent making a semi-premade solid phase suitable for use in a sandwich immunoassay.

[0809] 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 an exemplary flow chart of one non-limiting embodiment of a method 1200 in accordance with the present disclosure.

[0011] FSG. 4 depicts an exemplary Flow chart of another non-limiting embodiment of a method 1300 in accordance with the present disclosure,

[0012] FIG, 5 graphically depicts a Passing-Bablok regression (A) and Bland-Altman plot (B) for samples that were confirmed to have the TSH R75G mutation and were analyzed by the prior art TSH2 and newly developed total TSH assays on the ADVIA CENTAUR® XP system (Siemens Healthcare Diagnostics, Inc. Tarrytown, NY). The upper line in panel A is Passing-Bablok regression; the lower line is the line of identity.

[0013] 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 he beneficially incorporated in other embodiments without further recitation. DETAILED DESCRIPTION

[0014] 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 exempiary - 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.

[0015] 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 and procedures 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.

[0016] Al! 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.

[0017] AH 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. AH 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.

[0018] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0019] 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." [0S20] 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, 3,4, 5,10, 15, 2.0, 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. [0Q21] 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.

[0022] 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).

[0023] 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 ciaims.

[8024] 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 byway 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.

[8025] 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), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (such as, but not limited to, Fab, Fab', F(ab’)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. The antibody can be of any type or ciass (e.g., IgG, IgE, !gM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and !gA2).

[0026] "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 lightchain variable region domain in tight, non-covaient 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 ail 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.

[0027] By the term, "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DMA 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 beer? obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0026] 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 ail proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an "antigen" as that term is used herein. Furthermore, an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. 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.

[0029] 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, IgM, 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 immunoglobulin produced 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.

[0030] 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 polynucleotide,” as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g,, a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, 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. [0D31] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, “G" refers to guanosine, “T" refers to thymidine, and "U" refers to uridine. [0S32] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0033] 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-naturaliy 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.

[0034] 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, in certain non-limiting embodiments, the term refers to a variant as described in U.S. Patent Application Publication No. 2018 / 0222995.

[0035] 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 or state. 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, ora 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 ceils (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.). (QS36] As described herein, in some non■ limiting embodiments, a biomarker is a thyroidstimulating hormone or TSH Biomarker. A "TSH Biomarker" as used herein refers to a biological marker for thyroid or pituitary disorders. In some non-limiting embodiments, one or more TSH Biomarkers includeTSHB p.R75G variant (also referred to as "R55G,” based upon the Human Genome Assembly utilized, as described in detail in Example 2), or a combination thereof. In certain non-limiting embodiments, a TSH Biomarker includes a gene product associated with the specific recited biomarker. For example, depending on context, "TSH" refers to a nucleotide encoding TSH or a characteristic or functional fragment thereof, as well as a TSH protein or a characteristic or functional fragment thereof. In certain non-limiting embodiments, the TSH Biomarkers include variants of TSH including proteins having an amino acid sequence that has at least 90%, 95%, or 99% sequence identity to the wild type TSH or the beta subunit thereof (SEQ. ID NO:1), in certain non-limiting embodiments, a TSH Biomarker incudes a TSH hormone wherein the beta-subunit thereof has an alteration such as a substitution at position 75 (R75G; SEQ. ID NO:2), using SEQ. ID NO:1 for numbering.

[0037] The term "characteristic fragment" refers to a fragment of a biomarker (e.g., TSH 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 2.0, at least 30, at least 40, or at least 50 amino acids, in certain non-limiting embodiments, a characteristic fragment refers to a fragment of a biomarker that has at least 90%, at least 95%, or at least 99% sequence identity to the biomarker from which the characteristic fragment was derived. In certain non-limiting embodiments, SEQ. ID. NO:1 is a characteristic fragment of the TSH Biomarker. (0038] As used herein, the term "gene product" generally refers to an RNA transcribed from the gene (pre-and / or post-processing] or a polypeptide (pre- and / or post-modification) encoded by an RNA transcribed from the gene. [GG39] 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,      12SI, 1Z3I, &4Cu, 187Re, min, S0Y,"mTc, 177Lu, 89Z.r etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinium esters, 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 those described in U.S. Patent No. 6,664,043. In certain non-limiting embodiments, a suitable acridinium ester includes HEGAE described in U.S. Patent No. 6,664,043. (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, whethera 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 testingfora given disease, disorder, or condition.

[0041] As used in this specification and ciaim(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 demerits is not necessarily limited to only those elements but may include other elements not expressly listed or inherently present therein. [GG42] 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. [GG43] 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.

[0044] 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-covaient 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, [0Q45] 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 he 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.

[0046] 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 ul to about 100 qi, or a range of from about 1 pl to about 75 pl, or a range of from about 2 |il to about 60 pi, or a value less than or equal to about 50 pl, or the like.

[0047] The term "Triton X-1D0" as used herein refers to a nonionic surfactant that has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group. Triton X-100 may be referred to by the chemical names "octyiphenol ethoxylate," "polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether, "t-octylphenoxypolyethoxyethanol," and "polyethylene glycol tert-octylphenyl ether."

[0048] The term "TERGITOL™ 15-S-9" (Dow Chemical Company) as used herein refers to a nonionic surfactant that is a secondary alcohol ethoxylate. TERGITOL™ 15-S-9 may also be referred to by the name NP-40 as well as the chemical name "nonyl phenoxypolyethoxylethanoi."

[0049] The term "patient” as utilized herein includes human and veterinary subjects. In certain non-limiting embodiments, a patient Is a mammal. !n 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.

[0050] 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. [0Q51] The term "specific binding partner," as used in particular (but not by way of limitation) herein in the term "target anaiyte-specific binding partner," will be understood to refer to any molecule capable of specifically associating with the target analyte or target biomarker. For example, but not by way of limitation, the binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), com binations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte.

[8052] 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 an antibody (or 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 antibody (or fragment thereof) and the diagnostic biomarker. In certain non-limiting embodiments, the quantitative determination of such an immunocompiex 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 first antibody. Alternatively, the first version features a sandwich format in which the second antibody also binds a serum marker. 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.

[0053] In other particular (but non-limiting) embodiments, the immunoassays may detect a complex between a serum marker and a serum marker-binding antibody using a detection molecule (i.e., second reagent.) that is capable of binding to the serum marker-binding antibody and also capable of being detected when bound to the immunocompiex. For example (but not by way of limitation), the second reagent may include a label attached to a receptor, a ligand, or even another copy of the serum marker.

[0054] Turning now to particular non-limiting embodiments of the present disclosure, embodiments of the present disclosure include individual biomarkers forTSH, homologues, isoforms, and variants of TSH as well as compositions / kits / systems / devices / assays containing same, methods of producing and using same, and diagnostic tests related thereto. The identification of one or more biomarkers forTSH is attractive and advantageous for several reasons. First, the currently used diagnostic criteria forTSH may be limiting by failing to detect one or more variants of TSH. The identification of one or more serum biomarkers that are diagnostic forTSH will be helpful for both clinicians and patients, particularly in cases where a genetic background of an individual or population of individuals in need of testing produces a variant TSH that is poorly or undetectable in current assays.

[0055] The present disclosure is directed toward biomarkers, as well as compositions / reagents / kits / systems / devices / assays including reagents for measuring such biomarkers, along with methods of using same. In certain non-limiting embodiments, the biomarkers are wild type TSH and a variant of TSH. [0S56] In certain non-limiting embodiments, the present disclosure includes compositions, reagents, kits, systems, and methods for determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual. The method includes the steps of: (a) obtaining a biological fluid sample from an individual: (b) incubating the biological fluid sample with at least one antibody that specifically binds TSH and at least one antibody that binds to a TSH variant (such as, but not limited to, TSH R75G) under conditions that allow for formation of at least two antibody-TSH immunocompiexes (i.e., one immunocomplex containing wild type TSH and another immunocomplex containing TSH variant); (c) measuring an amount of antibody-TSH immunocompiexes formed to obtain a measured value for total TSH (wild type TSH + TSH variant) in the sample; and (d) using a mathematical algorithm to obtain a TSH score based on the measured value of total TSH Biomarker in the sample.

[0057] In certain non-limiting embodiments, the present disclosure includes compositions, reagents, kits, systems, and methods of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual. The method includes the steps of: (a) contacting a biological fluid sample from an individual with at least one antibody that specifically binds wild type TSH and at least one antibody that binds to a TSH variant (such as, but not limited to, TSH R75G) under conditions that allow for formation of at least two antibody-TSH immunocompiexes (i.e,, one immunocomplex containing wild type TSH and another immunocomplex containing TSH variant); (b) measuring an amount of antibody-TSH immunocompiexes formed to obtain a measured value for total TSH (wild type TSH + TSH variant) in the sample; and (c) using a mathematical algorithm to obtain a TSH score based on the measured value of total TSH Biomarker in the sample.

[0058] In certain particular (but non-limiting) embodiments, the at least two diagnostic markers include TSH and TSH variant, and wherein step (d) is further defined as combining the measured values of the two diagnostic markers using the mathematical algorithm to obtain the TSH score, in certain non-limiting embodiments, the TSH and TSH variant are each characterized as human.

[0059] In certain non-limiting embodiments, the present disclosure includes compositions, reagents, kits, systems, and methods of determining the presence, severity, and / or predisposition of disease in an individual. The method includes the steps of: (a) obtaining a biological fluid sample from an individual; (b) selecting at least two diagnostic markers of disease from said sample, wherein the at least two diagnostic markers are selected from the group consisting of TSH, TSH isoform, TSH variant (such as, but not limited to, R55G / R75G), or a polypeptide having 99% sequence identity to TSH, and combinations thereof; (c) measuring the amount of each of the at least two diagnostic markers in the sample to obtain a measured value for each of the at least two diagnostic markers; and (d) combining the measured values of the at least two diagnostic markers using a mathematical algorithm to obtain a TSH score (such as, but not limited to, a TSH score for thyroid or pituitary disease score). In certain non-limiting embodiments, the at least two diagnostic markers are TSH and a TSH variant characterized by R55G / R75G including substitution of R for G at position 55 / 75 (depending on the nomenclature of Human Genome Assembly utilized), in other non-limiting embodiments, the at least two diagnostic markers are TSH and a TSH variant having at least 99% sequence identity to wild type TSH.

[0060] In certain non-limiting embodiments, the present disclosure includes a kit for detecting thyroid disease, said kit including: (a) one or more anti-TSH Biomarker agents, wherein the one or more anti-TSH Biomarker agents include: (i) an anti-TSH agent, (ii) an anti-TSH variant agent: and (b) instructions for use. In certain non-limiting embodiments, the one or more anti-TSH Biomarker agents are man-made or synthetic. In certain non-limiting embodiments, the two or more anti-TSH agents comprise: a first antibody that specifically binds to a polypeptide comprising SEQ ID NQ:1: and a second antibody that specifically binds to a polypeptide comprising SEO. ID NO:2.

[0061] In certain non-limiting embodiments, the present disclosure relates to a non-transitory computer readable medium containing executable instructions that when executed causes a processor to perform operations comprising one or more of any of the embodiments described herein.

[0062] Also provided by the present disclosure are kits including one or more or two or more anti-TSH Biomarker agents and instructions for use (e.g., treatment, prophylactic, or diagnostic use), ’wherein one anti-TSH antibody specifically binds to wild type TSH and another anti-TSH antibody specifically binds to a variant of TSH. In some non-limiting embodiments, the kit is used for an in vitro diagnostic assay to diagnose thyroid disease, pituitary disorder, or combinations thereof. In some non-limiting embodiments, the one or more or two or more anti-TSH Biomarker agents include antibody agents. In some non-limiting embodiments, two or more of the antibody agents are labeled with a detectable moiety. In some non-limiting embodiments, the kit further includes a detection agent (e.g., one or more acridinium ester molecules), in some non-limiting embodiments, one or more of the antibody agents are labeled with one or more of the acridinium ester molecules such as (but not limited to) HEGAE. In some non-limiting embodiments, the kit further includes one or more secondary antibody agents that specifically bind to one or more of the anti-TSH Biomarker antibody agents.

[8063] In some non -limiting embodiments, the kit further includes one or mors control samples. In some non-limiting embodiments, the control samples include one or more TSH Biomarker standards.

[0864] In addition to the above, a kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, TERGITOL™ (Dow Chemical Company), 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-TSH Biomarker agents. In such embodiments, a kit can include instructions for admixing the anti-TSH Biomarker agents and the other ingredients, or for using the anti-TSH Biomarker together with the other ingredients.

[8065] 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, [0Q66] Also provided herein are compositions. In some non-limiting embodiments, a composition includes two or more of any of the anti-TSH Biomarker agents as described or otherwise contemplated herein. In some non-limiting embodiments, two or more TSH Biomarkers include anti-TSH Biomarker agents and anti-TSH variant biomarker agents, or a combination thereof. In certain non-limiting embodiments, the compositions include compositions shown in FIG. 1 individually, or collectively as a complex.

[0067] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing an immunoassay for thyroid-stimulating hormone (TSH). The kit includes a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigenbinding fragment thereof; a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or antigen-binding fragment thereof; a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof specifically binds to at least one variant ofTSH, and wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof Is conjugated to the second label; and a fourth reagent comprising a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label. The first and second anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of TSH so as to form a sandwich complex, and the antibody or antigen-binding fragment thereof of the fourth reagent specifically binds to the second label of the second reagent to attach the sandwich complex to the solid support; and wherein the first and third anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the TSH variant so as to form a sandwich complex, and the antibody or antigen-binding fragment, thereof of the fourth reagent binds to the second label of the third reagent to attach the sandwich complex to the solid support.

[0068] Certain non-limiting embodiments of the present disclosure are directed to a system for performing an immunoassay for TSH 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). [0Q69] Certain non-limiting embodiments of the present disclosure are directed to a method of determining a concentration of total TSH in a biological sample. The method includes one or more of the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigen-binding fragment thereof; (c) a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or antigen-binding fragment thereof; (d) a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or antigenbinding fragment thereof is conjugated to the second label: (e) a fourth reagent comprising a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label; (2) incubating the mixture under conditions whereby (b) and (c) bind to TSH present in the biological sample to form a first sandwich complex, (b) and (d) bind to TSH variant present in the biological sample to form a second sandwich complex, and the antibody of (e) binds to the second label to attach the first and second sandwich complexes to the solid support of (e); (3) detecting a signal generated by the first label indirectly bound to the solid support, of (e); and (4) determining a total concentration of TSH 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.

[0070] In a particular (but non-limiting) embodiment, the method is further defined as a method of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual.

[0071] In a particular (but non-limiting) embodiment, step (4) of the method is further defined as comprising the steps of: (i) measuring an amount of first and second sandwich complexes formed to obtain a measured value for total TSH (total TSH + TSH variant) in the biological sample; and (ii) using a mathematical algorithm (such as, but not limited to, discriminant function algorithm, i.e., a linear discriminant function algorithm) to obtain a TSH score based on the measured value of total TSH in the sample. The TSH score may be used (for example, but not by way of limitation) to support, predict, or substitute a thyroid test.

[0072] Any biological fluid sample known in the art or otherwise contemplated herein that may contain TSH or TSH variant that is detectaiale by two or more anti-TSH antibodies and that is indicative of thyroid disease or pituitary disorder 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).

[0073] Any anti-TSH antibodies known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Anti-TSH antibodies are well known in the art and commercially available. For example (but not by way of limitation), anti-TSH antibodies that may be utilized in accordance with the present disclosure as the antibodies bound to the first and second labels include those disclosed in US Patent Application Publication Nos. US2009 / 0087923; US 2009 / 0088334; US 2012 / 0301905; US 2012 / 0301906; US 2012 / 0301896: and US 2016 / 0109466; as well as US Patent Nos. 8,253,445; 8,617,826; 9,068,994; 9,199,234; and 9,575,077. In addition, anti-TSH monoclonal antibodies utiiizabie in accordance 'with the present disclosure are available from various commercial sources including (but not limited to) AbboMax (San Jose, CA); Abeam (Cambridge, UK); Anogen (Mississauga, Ontario); Antibodies-Online Inc. (Limerick, PA); Aviva Systems Biology (San Diego, CA); BBi Solutions (Portland, ME); Biocare Medical (Concord, CA); Biorbyt (Cambridge, UK); Bioss Inc. (Woburn, MA); BosterBio (Pleasanton, CA); Creative Biolabs, Inc. (Shirley, NY); Creative Diagnostics (Shirley, NY); Fitzgerald Industries (Gardner, MA); GeneTex (Irvine, CA); GenScript (Piscataway, NJ); invitrogen (Waltham, MA); Medix Biochemica (St. Louis, MO); LifeSpan Biosciences Inc. (Lynnwood, WA); MyBioSource, Inc. (San Diego, CA); Novus Biologicals (Littleton, CO); OriGene Technologies (Rockville, MD)); Santa Cruz Biotechnology, Inc. (Dallas, TX); ThermoFisher Scientific (Waltham, MIA); US Biological Life Sciences (Salem, MA); 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 dearly 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. [GG74] in certain particular (but non-limiting) embodiments, anti-TSH mouse monoclonal antibodies are suitable for use herein, such as (but not limited to) those produced by Fitzgerald Industries (Gardner, MA). In certain other particular (but non-limiting) embodiments, FITC antibodies are suitable for use herein including FITC-mAB5405 obtainable from Medix Biochemica (St. Louis, MO) and FITC-MAB130P obtainable from BBI Solutions (Portland, ME). For example, Table 1 from Medix Biochemica demonstrates how the commercially available anti-TSH antibodies 5401, 5404, 5405, 5407, 5408, and 5409 can be utilized in combination in a sandwich immunoassay. TABLE 1 DETECTION ANTIBODY 5401 5404 5405 5407 5408 5409 5401 - 4™ 4- - -b a o 5404 - -b -b ■b ■— 5405 - - - -b LU cr o 5407 - - - - + -b < 5408 -b -b - -b 5409 - ■b 4- - [0Q75] In addition, non-limiting examples of antibodies that recognize the TSH variant R55G / R75G include MAB130P (and in particular, but not by way of limitation, HTC-MAB130P) from BBI Solutions (Portland, ME) and the antibodies disclosed in US Patent Application Publication No. 2025 / 0044304.

[0076] 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-TSH 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-TSH antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-TSH antibodies or the characteristics thereof is deemed necessary.

[0077] in certain non-limiting embodiments, a suitable TSH biomarker for use herein (and to which an antibody utilized in accordance with the present disclosure can specifically bind) includes a polypeptide comprising the amino acid sequence: MTALFLMSMLFGLTCGQAMSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQD VCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYLVGFS (SEQ ID NO:1), or fragments or variants thereof, e.g., variants having at least 95%, at least 97%, or at least 99% sequence identity to this biomarker sequence.

[0078] In certain non-limiting embodiments, a suitable biomarker for use herein (and to which an antibody utilized in accordance with the present disclosure can specifically bind) includes a polypeptide comprising the amino acid sequence: MTALFLMSMLFGLTCGQAMSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQD VCTYGDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYLVGFSV (SEQ ID NO:2), or fragments or variants thereof, e.g., variants having at least 95%, at least 97%, or at least 99% sequence identity to this biomarker. Note that SEQ ID NO:2 is the R55G / R75G variant of TSH.

[0079] 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, FASTA, L-AUGN, etc.), available through the worldwide web at sites including GENBANK (National Center 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.

[0880] Any of the antibodies or antigen-binding fragments described or otherwise contemplated herein may be labeled or otherwise conjugated to various chemical or biomoiecule 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 (H) chelates), cloned enzyme donors, photosensitizer particles or chemiluminescer particles tor 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, fodine-123, iodine-124, iodine-125, bromine-77, indium-Ill, 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 he conjugated to the described antibodies forthe 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,

[0081] 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 limited to, 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.

[0082] 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).

[0083] 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 an antibody or other specific binding partner is available that can be conjugated to the solid support, so long as the second label and the antibody / binding partner do not otherwise interfere with the assay being performed. Non-limiting examples of second labels and antibody / specific binding partner combinations that can be utilized in the kits, systems, and methods in accordance with the present disclosure include fluorescein (e.g., fluorescein isothiocyanate (FITC)) for binding to anti-fluorescein antibody or fragment thereof; biotin for binding to avidin, streptavidin, or anti-biotin antibody; digoxigenin for binding to anti-digoxigenin antibody; other hapten and binding partner combinations; and the like. These types of label and antibody / specific binding partner combinations are well known in the art and widely available commercially. Thus, no further description thereof is deemed necessary.

[0084] 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, N.J.); 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 paramagnetic particles, some source of a magneticfield may be used to retain the particles and molecules bound directly or indirectly to the particles during an optional wash step. The molecules may be bound covalently, by salt-bridges, hydrogen bonding, or another type of bond.

[0085] 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.

[0086] 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. [Q0§7] In a particular (but non-limiting) embodiment, the reagent comprising the anti-TSH variant antibody conjugated to the second label and the reagent comprising the solid support coated with the anti-second label antibody are combined into a single component / compartment within the kit.

[0088] 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.

[0089] According to European Union REACH regulations, Triton X-100 nonionic surfactant is considered a "Substance of Very High Concern" (SVHC), and therefore its use and sale within the European Union is banned unless a specific authorization is granted. Therefore, if? certain non-limiting embodiments, ail of the reagents utilized in accordance with the present disclosure are free of Triton X-100 and instead contain another nonionic surfactant that is not subject to REACH regulations, such as (but not limited to) TERGITOL™ 15-5-9 (Dow Chemical Company).

[0090] In certain non-limiting embodiments, the present disclosure facilitates point of care or remote diagnoses of thyroid disease or pituitary disorder and assists health care providers in monitoring the status or progress of thyroid disease or pituitary disorder (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.

[0091] In certain non-limiting embodiments, the present disclosure employs computer-implementable algorithmic methods which utilize one or more TSH-reiated marker values. The predictive value of the present disclosure is validated in clinical studies to monitor the status or progress of thyroid disease or pituitary disorder.

[0092] In certain non-limiting embodiments, analytical methodology is applied to information obtained from a TSH assay 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.

[0093] 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 non linear function algorithm derived in a manner described herein.

[0094] 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 serum marker values.

[0095] Methods and kits provided herein are able to detect TSH 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 detection and / or diagnosis of thyroid disease in a subject detects TSH or variants thereof 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 nonlimiting embodiments, methods and kits provided herein can detect TSH or variants thereof with a sensitivity 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 TSH ar variants thereof 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%.

[0096] 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 TSH Biomarker score based on receiving a TSH Biomarker profile and / or a level of two or more of any of the TSH 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.

[0097] 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., thyroid disease tests), identify a subject as being at risk of or having thyroid disease or pituitary disorder, and / or identify a subject to receive medication based on received data (e.g., a TSH Biomarker profile) and use the data to calculate a TSH Biomarker score. A system may be able to rankorder identified next steps based on a TSH 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 thyroid disease.

[0098] 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.

[0099] 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 processing instructions stored in the memory 1120 or on the storage device 1130, including for receiving or sending information through the input / output device 1140.

[0100] 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, [GW1] 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.

[0102] 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.

[0103] 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 a method as provided herein. For example, a non-transitory computer readable medium containing executable instructions that tvhen executed cause a processor to perform operations including a method described herein are shown in FIG. 3 and FIG. 4, In certain non-limiting embodiments, a non-transitory computer readable medium includes a hard drive, external hard drive, discs, CDs, DVDs, ar?d the like that stores data. In certain non-limiting embodiments, software disposed within a physical medium is suitable for use herein.

[0104] 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, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual is disclosed. The method includes the steps of: [a) incubating a biological fluid sample with two or more antibodies that specifically bind to TSH Biomarkers (at least one to wild type TSH and at least one to a TSH variant) under conditions that allow for formation of at least two antibody-TSH immunocomplexes; (b) measuring an amount of antibody-TSH immunocomplexes formed to obtain a measured value forTSH in the sample; and (c) using a mathematical algorithm to obtain a TSH score based on the measured value of TSH in the sample, in certain non-limiting embodiments, incubating a biological fluid sample includes incubating with two or more antibodies that specifically binds to TSH and a highly related TSH variant under conditions that allow for formation of an antibody-TSH immunocomplex.

[0105] Referring now to FIG. 3, said Figure depicts a method 1200 of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual in accordance with the present disclosure. [01G6] Referring now to FIG. 4, said Figure depicts a method 1300 of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual in accordance with the present disclosure. EXAMPLES

[0107] Exampies 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 is meant to be exemplary, not exhaustive. Example 1 [0W8J FIG. 1 graphically depicts one non-limiting embodiment of a total TSH assay constructed in accordance with the present disclosure and that detects both wild type TSH (WT TSH) as well as the TSH variant R75G in a biological sample. The assay utilizes the following components: (i) an Ancillary Well reagent that comprises a HTC-labeled mouse monoclonal antibody that specifically binds to WTTSH (FITC-mAB5405); (ii) a solid phase reagent that comprises paramagnetic particles coated with an anti-FITC antibody (PMP::anti-FITC), and to which has been added a FITC-iabeled antibody that detects TSH R75G (FITC-mAB130P); and (iii) a Ute reagent that comprises a Fab fragment of another a nti-TSH antibody that binds to both WT TSH and TSH R75G and is labeled with an acridinium ester (Fab54Q9::BSA::HEGAE),

[0109] The assay is also performed in the presence of TERGITOL™ 15-S-9 (Dow Chemical Company) at 3 g / L. [QUO] In one non-limiting embodiment of the assay method, 100 pL of biological sample is combined with 50 pL of Ancillary Well reagent and 50 pl of Lite reagent and incubated for about 2.75 minutes. Then 200 pL of the Solid Phase reagent is added, and the mixture is incubated for 5,5 minutes. Then a wash step is performed, and Acsd and Base reagents are added to initiate the chemiluminescent reaction; the amount of relative light units (RLUs) generated are subsequently detected by the system. Example 2 [Gill] A new and improved TSH assay and kit are provided for in vitro diagnostic use in the quantitative determination of thyroid-stimulating hormone (TSH, thyrotropin) in biological samples, such as (but not limited to) human serum and plasma (EDTA and lithium heparin) using a clinical analyzer, such as (but not limited to) the ATELLICA® IM Analyzer (Siemens Healthcare Diagnostics, Inc. Tarrytown, NY). Measurements of thyroid stimulating hormone produced by the anterior pituitary are used in the diagnosis of thyroid or pituitary disorders.

[0112] Thyroid-stimulating hormone is a glycoprotein with two non-covalently bound subunits. The alpha subunit is similar to those of follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and luteinizing hormone (LH). The beta subunit of TSH is unique, which results in the specific biochemical and immunological properties of this hormone. TSH is synthesized and secreted by the anterior pituitary in response to a negative feedback mechanism involving concentrations of FT? (free T?) and FT^ (free T«). Additionally, the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), directly stimulates TSH production, TSH interacts with specific ceil receptors on the thyroid ceil surface and exerts two main actions: the first action is to stimulate cell reproduction and hypertrophy; the second action is to stimulate the thyroid gland to synthesize and secrete Ta and T4.

[0113] The ability to quantitate circulating levels of TSH is important in evaluating thyroid function. It is especially useful in the differential diagnosis of primary (thyroid) hypothyroidism from secondary (pituitary) hypothyroidism and tertiary (hypothalamus) hypothyroidism, in primary hypothyroidism, TSH levels are significantly elevated, while in secondary and tertiary hypothyroidism, TSH levels are low. TRH stimulation differentiates secondary and tertiary hypothyroidism by observing the change in patient TSH levels. Typically, the TSH response to TRH stimulation is absent in cases of secondary hypothyroidism, and normal to exaggerated in tertiary hypothyroidism. Historically, TRH stimulation has been used to confirm primary hyperthyroidism, indicated by elevated T? and T4 levels and low or undetectable TSH levels. TSH assays with increased sensitivity and specificity provide a primary diagnostic tool to differentiate hyperthyroid from euthyroid patients.

[0114] In one non-limiting embodiment, this assay employs a FITC-labeied mouse monoclonal anti-wild type TSH antibody, a FITC-labeied mouse monoclonal anti-TSH variant antibody, mouse monoclonal anti-fluorescein antibody linked to paramagnetic particles, and a tracer including an acridinium ester (AE)-labeled antl-TSH mouse monoclonal antibody conjugated to bovine serum albumin (BSA) for chemiluminescent detection, wherein this AE- labeled antibody can bind to both TSH and TSH variant, whereby toe binding of either of the FITC-labeled antibodies and binding of the AE-labeied antibody results in the formation of a sandwich immunocompiex (one sandwich immunocompiex with TSH and another sandwich immunocompiex with TSH variant), A direct relationship exists between the amount of TSH present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0115] This assay may be utilized with any biological specimens, such as (but not limited to) serum and plasma (EDTA and lithium heparin), in a particular (but non-limiting) embodiment, this assay requires at least 75 pi. of sample for a single determination; this volume does not include the unusable volume in the sample container or the additional volume required when performing duplicates or other tests on the same sample. Do not use samples with apparent contamination. Before placing samples on the system, ensure that samples are free of bubbles, foam, fibrin, or other particulate matter. Remove particulates by centrifugation according to CLSi guidance and the collection device manufacturer's recommendations.

[0116] In one non-limiting embodiment, the Materials present in the primary reagent pack kit include: « Lite reagent (6.00 mL / reagent pack) - BSA conjugated to mouse monoclonal anti-TSH antibody (~0.3 pg / mL) labeled with acridinium ester in buffer mouse IgG; BSA; Bovine Gamma Globulin (BGG); goat serum; sodium azide (<0.1%); surfactant; and preservatives. • Solid Phase (21.0 mL / reagent pack) - FITC-labeled mouse monoclonal anti-TSH variant antibody and mouse monoclonal anti-fluorescein antibody linked to paramagnetic particles (85 p.g / rnL) in buffer; BSA; BGG; goat serum; sodium azide (<0.1%); surfactant; preservatives. The anti-TSH variant antibody binds to at least one variant, such as (but not limited to) the R55G / R75G TSHB variant. « Ancillary Weil Reagent (6.D mL / reagent pack) -- FITC-labeled mouse monoclonal antiwild type TSH antibody (~3 qg / mL); BSA; BGG; goat serum; sodium azide (<0.1%); surfactant; preservatives. In this manner, the assays and kits of the present disclosure are capable of accurately measuring TSH levels in rare genetic variant populations.

[0117] The kits of the present disclosure may further include at least one calibrator that contains a known concentration of TSH. In certain particular (but non-limiting) embodiments, the kits may include a "low" calibrator and a "high" calibrator, wherein the low calibrator has a lower concentration of TSH than the high calibrator. The calibrator may contain wild type TSH and / or a TSH variant.

[0118] In one non-limiting embodiment, the assays and kits disclosed herein are utilized on a clinical analyzer system that performs the following steps (either automatically or manually): 1. Dispenses 75 pl. of sample into a cuvette. 2. Dispenses 38 uL of Ancillary Well Reagent and 38 pl of Lite Reagent, then incubates for 5 minutes at 37“C. 3. Dispenses 150 pL of Solid Phase, then incubates for 7 minutes at 37X. 4. Performs a wash sequence using a wash reagent (such as, but not limited to, ATELLICA® IM Wash, Siemens Healthcare Diagnostics, Inc. Tarrytown, NY)). 5, Dispenses 300 pL each of acid and base reagents (such as, but not limited to, ATELLICA® IM Acid Reagent and ATELLICA® IM Base Reagent) to initiate the chemiluminescent reaction. 6. Reports results.

[0119] Ths system determines the result using the calculation procedure described in the system online help. The system reports results in plU / mL (common units) or mIU / L (51 units), depending on the units defined when setting up the assay. The conversion formula is 1 plU / ml. - 1 mIU / L. Results of this assay should always be interpreted in conjunction with the patient's medical history, clinical presentation, and other findings, if any.

[0120] Expected values were established using the ADVIA CENTAUR® prior art TSH3-UL assay on the ADVIA CENTAUR® system (Siemens Healthcare Diagnostics, Inc. Tarrytown, NY) and confirmed by assay comparison. The reagent formulations used on the ATELLICA® IM Analyzer (Siemens Healthcare Diagnostics, Inc. Tarrytown, NY) are the same as those used on the ADVIA CENTAUR® systems.

[0121] A reference interval for healthy (euthyroid) adults was established using the ADVIA CENTAUR® prior art TSH3-UL assay on the ADVIA CENTAUR® system and verified using the ATELLICA.® IM new total TSH assay on the ATELLICA® IM Analyzer in accordance with CLSI Document EP28-A3C.

[0122] Samples were assayed for TSH, FTs, and FT4 and considered normal if their values were within acceptable ranges. Samples were also screened for the presence of thyroid autoantibodies (Kratsch et al. (2008) Ciin Biochem, 41(13):1091-1098). The reference interval is shown in Table 2 and was determined by calculating the 2.5th and 97.5th percentiles of the distribution of values. TABLE 2 Group Na Reference Interval ulU / mL (mIU / L) Euthyroid Adults 229 0.55-4.78 a Number of samples tested.

[0123] Reference intervals for healthy pediatric population (infants, children, and adolescents) were established in accordance with CLSi Document EP28-A3C using the ADVIA CENTAUR* prior art TSH3-UL assay on the ADVIA CENTAUR® system.

[0124] Samples were collected prospectively from apparently healthy (euthyroid) pediatric subjects, using predefined inclusion criteria. A non-parametric approach was used to establish the reference intervals for children and adolescents where the 2.5th and 97.5th percentiles of the distribution of values were calculated. For the infant population, the reference interval was calculated using an approach to accommodate the smaller sample size.

[0125] Performance Characteristics: The reagent formulations used on the ATELLICA® IM Analyzer are the same as those used on the ADVIA CENTAUR® systems. Some performance characteristics for the ATELUCA® IM assay were established using the ADVIA CENTAUR® systems.

[0126] The measuring interval was 0,010-150.000 plU / ml. (mIU / L). The lower limit of the measuring interval is defined by the limit of quantitation (LoQ). Results below the measuring interval are reported as < 0.010 plU / mL (miU / L).

[0127] The detection capability includes the following values: Limit of Blank (LoB) ■■ 0.005 plU / ml. (mIU / L); Limit of Detection (LoD) - 0,008 piU / mL (mIU / L); and Limit of Quantitation (LoQ) - 0.010 piU / mL (mIU / L). Detection capability was determined in accordance with CLSi Document EP17-A2, The LoB corresponds to the highest measurement result likely to be observed for a blank sample with a probability of 95%, The LoD corresponds to the lowest analyte concentration that can be detected with a probability of 95%, The LoQ corresponds to the lowest analyte concentration at which the within laboratory CV is < 20%.

[0128] Precision was determined in accordance with CIS! Document EP05-A3. Samples were assayed in replicates of two with two runs per day using a 20-day protocol.

[0129] The results shown in Table 3 is one representative of the performance of the assay. The assay was designed to have the precision shown in Table 4 when using a 20-day protocol in accordance with CIS! Document EP05-A3. TABLE 3 Sample N’ Mean plU / ml (mIU / L) Repeatability Within-Laboratory Precision SDb plU / mL (mlU / L) CVC (%) SD UlU / mL (mIU / L) CV (%) Serum A 80 0.086 0.0018 2.1 0.0027 3.2 Serum 8 80 0.194 0.0043 2.2 0.0050 2.6 Serum C 80 0.496 0.0103 2.1 0.0156 3.1 Serum D 80 4.739 0.0974 2.1 0.1328 2.8 Serum E 80 48.040 1.2643 2.6 1.7163 3.6 Serum F 80 98.207 2.4514 2.5 2.8295 2.9 Plasma, lithium heparin A 80 0.096 0.0018 1.8 0.0030 3.1 Plasma, lithium heparin B 80 0.513 0.0112 2.2 0.0168 3.3 Plasma, lithium heparin C 80 4.802 0.1108 2.3 0.1700 3.5 Plasma, lithium heparin D 80 52.769 1.1208 2.1 1.4833 2.8 Plasma, lithium heparin E 80 93.097 3.1793 3.4 4.2404 4.6 Control 1 80 0.103 0.0016 1.6 0.0022 2.2 Control 2 80 0.522 0.0071 1.4 0.0121 2.3 Control 3 80 4,800 0.0505 1.1 0.1173 2.4 Control 4 80 47.803 0.7748 1.6 1.0919 2.3 Control 5 80 101.770 2.1680 2.1 2.9022 2.9 8 Number of measu rements. b Standard deviation. c Coefficient of variation. TABLE 4 Concentration Interval Precision Repeatability (Within-Run) Within-Laboratory (Total Precision) 0.020-0.299 plll / mL (mIU / L) s12%CV s16%CV > 0.300-90.000 pltl / mL (mIU / L) <6%CV <8%CV >90.000 plU / mL (mIU / L) <7%CV <10% CV

[0130] Reproducibiiity was determined in accordance with CLSi Document EP05-A3. Testing was performed using three instruments and three reagent lots. Samples were assayed in replicates of five with one run per day using a 5-day protocol (225 measurements per sample).

[0131] The resuits shown in Table 5 are representative of the performance of the assay. The assay was designed to have the reproducibility shown in Table 6 when using a 5-day protocol in accordance with CLSi Document EP05-A3. TABLE 5 Sample Mean plU / mL (mIU / L) Repeatability Between Day Between Lot Between Instrument Reproducibility SD® plUImL (mIU / L) CV” SD plU / mL (mlU / L) cv W SD pltl / mL (mIU / L) cv W SD plWmL (mIU / L) cv W SD plU / mL (mIU / L) 2 g Serum A 0.087 0.002 2.12 0.001 1.30 0.003 3.44 0.001 1.26 0.004 4.43 Serum B 0.172 0.004 2.12 0.002 1.42 0.005 2.82 0.002 0.92 0.007 3.91 Serum C 0.449 0.009 2.04 0.004 0.92 0.011 2.52 0.003 0.58 0.015 3.42 Serum D 4.450 0.094 2.12 0.054 1.21 0.067 1.S0 0.044 0.98 0.135 3.03 Serum E 53.797 1.160 2.16 0.672 1.25 1.042 1.94 0.614 1.14 1.805 3.36 Serum F 100.021 2.250 2.25 1.789 1.79 3.066 3.06 0.590 0.59 4.244 4.24 Plasma A 0.090 0.002 2.23 0.001 1.07 0.003 3.54 0.002 1.86 0.004 4.70 Plasma 8 0.422 0.009 2.13 0.004 1.06 0.010 2.42 0.007 1.54 0.016 3.73 Plasma C 4.293 0.080 1.86 0.082 1.91 0.065 1.52 0.023 0.54 0.134 3.12 Plasma D 47.836 1.052 2.20 0.789 1.65 0.768 1.61 0.731 1.53 1.689 3.53 Plasma E 98.087 2.139 2.18 2.512 2.56 2.877 2.93 1.824 1.86 4.743 4.84 Control 1 0.102 0.002 2.06 0.001 1.12 0.004 3.71 0.001 0.69 0.005 4.44 Control? 0.506 0.008 1.63 0.005 1.08 0.013 2.57 0.003 0.59 0.017 3.28 Control 3 4.627 0.082 1.78 0.045 0.96 0.079 1.70 0.024 0.52 0.125 2.69 Control 4 45.957 0.854 1.86 0.586 1.28 0.725 1.58 0.000 0.00 1.264 2.75 Control 5 97.579 2.007 2.06 1.228 1.26 2.508 2.57 0.000 0.00 3.439 3.52 a Standard deviation. b Coefficient of variation. TABLE 6 Concentration Interval Reproducibility 0.020-0.299 plU / mL (miU / L) s;18.5% CV a 0.300-90.000 plU / ml (mIU / L) sia.5%CV > 90.000 pMmL (mIU / L) s12.5%CV

[0132] Assay comparison was determined with the Passing-Babiok regression mode! in accordance with CLSi Document EP09c-ed3. The resuits are shown in Table 7, Agreement of the assays may vary depending on the study design, comparative assay, and population tested. The assay is designed to have a correlation coefficient of > 0.97 and a slope of 1.0 ± 0.075. TABLE 7 Sample Comparative Assay (x)              Regression Equation Sample Interval     Na i* Serum ADVIA Centaur TSH3ULII assay using y «0.96x-0.010 plU / mL 0.011-144.5 plUfmt 323 0.999 the ADVIA Centaur XP system 3 Number of samples tested. b Correlation coefficient.

[0133] Specimen equivalency was determined with the Passing-Babiok regression mode! using the ADVIA CENTAUR® XP system in accordance with CLSI Document EP09c-ed3. The results are shown in Table 8. Agreement of the specimen types may vary depending on the study design and population tested. The assay is designed to have a correlation coefficient of > 0.95 and a slope of 0.95 ± 1.05. TABLE 8 Tube (y) vs. Serum (x)        Regression Equation          Sample Interval Na rb        I Plasma, EDTA                y = 0.99x - 0.019 plU / mL        0.050-147.805 plUfmL 52 0.999 | Plasma, lithium heparin       y = 1.01x-0.034 plU / mL        0.115-135.881 plUfmL 57   0.990 | 3 Number of samples tested. b Correlation coefficient.

[0134] Linearity testing was performed in accordance with CLSI Document EP06-ed2. The assay was linear for the measuring interval of 0.010-150.000 plU / mL (mIU / L).

[0135] High TSH concentrations can cause a paradoxical decrease in the RLUs (high-dose hook effect). In this assay, patient samples with TSH concentrations above the measuring interval and as high as 3000 plU / ml. will report > 150p!U / ml (mIU / L).

[8136] The assay standardization is traceable to the World Health Organization (WHO) 3rd International Reference Preparation for human TSH (IRP 81 / 565). Exampie 3

[0137] Summary of Background: Discordant resuits from thyroid-stimulating hormone (TSH) assays from various manufacturers have been reported for patients with an R75G mutation in the TSH 3 chain. Although functional, TSH with this mutation (TSH R75G) displays altered immunoreactivity, resulting in a lack of its detection by some assays, such as (hut not limited to) Siemens Healthineers' TSHS-Ultra (TSH3 UL) assay. The new total TSH assay of this Example was developed to address this issue. A method comparison study was conducted with the developed assay and a reference assay, i.e., Siemens Healthineers' TSH assay, which accurately measures TSH R75G.

[0138] Summary of Methods: Fifty-nine native human serum samples containing the TSH R75G mutation from two laboratories in India and the United States were analyzed. PCR and DNA sequencing confirmed R75G mutations in all samples. To measure TSH R75G, two replicates per sample were analyzed by the prior art TSH assay and the new total TSH assay.

[0139] Summary of Results: Mean TSH concentrations measured by the new total TSH assay ranged from 0.874-23.209 piU / mL. This range is within the assay's measuring interval (0.010-150.000 plU / ml) and is comparable to mean concentrations measured by the prior art TSH assay (range, 0.850-22.536 plU / mL). Concentrations of TSH P.75G in individual samples measured by the new total TSH assay were similar to those measured by the prior art TSH assay. Fifty-three samples had TSH concentrations <10 plU / mL; six had TSH concentrations >10 plU / rnL. Passing-Bablok regression analysis indicated the new total TSH assay performed comparably to the prior art TSH assay. A Bland-Altman plot provided confirmatory results.

[0140] Summary of Conclusions: The assays of the present disclosure can accurately and precisely measure TSH R75G in native human serum.

[0141] Introduction

[0142] Measurement of circulating thyroid-stimulating hormone (TSH) concentrations is crucial for the investigation of thyroid dysfunction, monitoring levothyroxine therapy, and monitoring therapy for hyperthyroidism (Van Uytfanghe, etal. (2023) Thyroid®. 33(9):1013-1028). Inaccurate readings by a TSH assay can lead to inaccurate diagnosis and / or inappropriate treatment.

[0143] Previous reports have described discordant TSH assay results for patients with the R75G mutation in the 3 chain of TSH (TSH R75G), which is also known as the R55G mutation (the R55G nomenclature is based on Human Genome Assembly GRCh37.pl3: chrl:115,576,654 A > G (2013), and the R75G nomenclature is based on the updated Human Genome Assembly GRCn38.pl4: chrl:115,034,033 A > G) (2022). See Genome Reference Consortium. Human Genome Overview - GRCh37.pl3, 2013; and Genome Reference Consortium. Human Genome Overview - GRCh38.pl4,2022. Some manufacturers'TSH assays were able to accurately measure concentrations of TSH R75G, whereas particular Siemens Healthineers' TSH assays on multiple instrument platforms were unable to detect and measure TSH with this mutation (Drees, et al., J Clin Endocrinol Metab. 2014;99(4):1171 1179; Pappa, et al., Thyroid. 2015;25(8);869-876; Rahimkhani, et al., Thyroid Res. 2020;13:2; and Shaki, et al. Eur Thyroid J. 2022;ll(l)). Further investigation showed that TSH R75G is functional, but its immunoreactivity is altered, resulting in a lack of detection by the solidphase monoclonal antibody (mAb) in the Siemens Healthineers' TSH3-Ultra (TSH3 UL) assay (Drees et al., incorporated supra).

[0144] To address the inability of the TSH3-UL assay to detect TSH R7SG, the assays of the present disclosure were developed. Unlike its predecessors, the assays disclosed herein contain an additional mAb to detect TSH R75G. This Example reports the results of a method comparison study of measurement of TSH R75G by the new assay and a reference method, i.e., the Siemens Healthineers' prior art TSH assay (which utilizes a polyclonal capture antibody), which has been previously shown to accurately measure TSH R75G (Drees et al., incorporated supra).

[0145] Materials and Methods

[0146] This method comparison study was performed in accordance with CIS! EP09c-ED3 (CLSI. Measurement Procedure Comparison and Bias Estimation Using Patient Samples. 3rd ed. CLSI guideline EP09c. Wayne, PA: Clinical and Laboratory Standards Institute; 2018). The two TSH assays differed in that the prior art TSH assay (Siemens Healthineers, Inc.) has a solidphase sheep polyclonal antibody to TSH and a lite-reagent mouse mAb to TSH, whereas the current assay uses a fluorescein isothiocyanate (FITC)-labeled mouse mAb to TSH variant, a FITC-labeled anti-wild type TSH capture mouse mAh, and mouse anti-fluorescein antibody linked to paramagnetic particles, and a tracer consisting of a proprietary acridinium ester and an anti-TSH mouse mAb conjugated to bovine serum albumin for chemiluminescent detection.

[0147] Seventy-four native human serum samples that contained TSH R75G were generously provided by Dr. Julia Drees (the Kaiser Permanents Northern California Regional Laboratory, Richmond, CA) and Dr. Nimrni Kansal (Dr. Lal PathLabs, Delhi, India). These samples were chosen based on prior analyses that showed the TSH results were below the limit of quantitation (LoOj of the prior art TSH3-UL assay analyzed with ADVIA CENTAUR® XP or ATELLICA® IM hut were above the LoQ of other prior art TSH assays, including the prior art TSH assay that utilizes polyclonal antibodies. AH samples were received under a Material Transfer Agreement (MTA) between Siemens Healthineers and the respective laboratories.

[0148] The presence of the R75G mutation was confirmed by PCR and sequencing of DNA (Antibody Systems, Inc., Hurst, TX USA). DNA was extracted from clotted blood from Dr. Lal PathLabs by using a QiAamp DNA blood mini kit (QIAGEN, Germantown, MD) according to the manufacturer's protocol. PCR amplification was performed using a Phusion High Fidelity PCR kit (Thermo Fisher Scientific lnc„ Waltham, MA) and TSWS-specifk forward (5'-TGTTTCCTAAAGTCCTGTCAC-3'; SEQ ID NO:3) and reverse (S'-GCTTTATTTCAGGCAAGCAC-3'; SEO, ID NO:4) primers (IDT Technologies, Coralville, (A), Products underwent Sanger sequencing (GENEWIZ, South Plainfield, NJ) using the same primers as those for amplification. The Human Genome Assembly GRCh38.pl4 was used as a reference genome, and position chrl:115,034,033 served as a reference point for the mutation. Sequences were analyzed using SnapGene version 6.2.1 (GENEWIZ). For all samples from the Drees laboratory, the R75G mutation had been confirmed earlier by PCR and DNA sequencing (Drees et al., incorporated supra).

[0149] Samples underwent testing for detection and measurement of TSH R75G by the ADVIA CENTAUR* XP TSH assay and the new total TSH assay format. Two replicates of each sample were tested in accordance ’with the manufacturer's instructions with each TSH assay, kit-specific calibrators, standards, and a commercial control (Bio-Rad Lyphochek Immunoassay Pius Control, Hercules, CA) on the ADVIA CENTAUR® XP system. Data reduction and analysis were performed with Raven Calculation software version 4 (Siemens Healthineers Diagnostics, Tarrytown, New York), and a one-sided 95% confidence interval with Passing-Sablok linear regression was used.

[0150] Results and Discussion

[0151] Of the 74 native serum samples with confirmed TSH R75G, 15 were excluded from further analysis because of insufficient volume for two replicates for each assay. Mean TSH concentrations in the remaining 59 samples ranged from 0.874-23.209 |iiU / mL (mIU / L) as measured by the new total TSH assay (Table 9). This range is within the measuring interval for this assay (0.010-150.000 ulU / ml [mIU / L]) and is comparable to that measured by the prior art TSH assay that utilizes polyclonal antibodies (mean, 0.850-22.536 piU / mL [mIU / L]).

[0152] Comparison of individual samples showed that the concentrations of TSH R75G measured by the new total TSH assay were similar to those measured by the reference TSH assay that utilizes polyclonal antibodies (Table 9). Most sample concentrations were <10 plU / mL (TSH concentration <2 plU / ml for 21 samples; 2-5 plU / ml for 24 samples; 5-10.0 piU / ml for 8 samples), and six samples had TSH concentrations >10 plU / ml. TABLE 9 Sample3 TSH R75G concentrations, plU / mL (mIU / L) New Total TSH assay Old TSH assay Replicate 1 Replicate 2 Replicate 1 Replicate 2 DSaml 4.216 4.363 4.295 4.692 DSamS 2.037 2.050 2.038 1.934 DSam8 1.759 1.827 1.470 1.425 DSamlO 1.984 1.944 1.656 1.623 DSamll 1.284 1.255 1.177 1.329 DSaml2 1.752 1.731 1.592 1.624 DSaml4 1.923 1.935 1.694 1.824 DSaml5 2.317 2.305 2.628 2.536 DSaml6 1.547 1.659 1.836 1.778 DSaml7 1.585 1.540 1.603 1.529 DSaml8 1.414 1.412 1.366 1.436 DSaml9 1.208 1.327 1.485 1.406 DSam20 0.984 0.949 1.073 1.097 DSam21 6.436 6.376 6.462 6.251 DSam22 3.667 3.950 4.027 4.135 DSam23 12.010 11.993 12.792 11.751 LSaml 2.407 2.508 2.587 2.571 LSam2 2.492 2.418 2.257 1.987 LSam3 2.057 2.063 1.770 2.008 LSam4 1.862 1.926 1.652 1.808 LSam5 3.301 3.243 3.296 3.272 LSam6 1.774 1.791 1.964 2.097 LSam7 6.520 6.878 7.004 6.459 LSam8b 7.883 7.924 7.640 7.842 LSamll 5.026 5.086 5.222 5.428 LSaml2 2.422 2.394 2.568 2.442 Sample3 TSH R75G concentrations, plU / mL (mIU / L) New Total TSH assay Old TSH assay Replicate 1 Replicate 2 Replicate 1 Replicate 2 LSaml4 6.483 6.528 6.101 5.906 LSaml5 2.528 2.534 2.429 2.200 LSaml6 3.610 3.644 3.409 3.585 LSaml8 4.794 4.694 4.884 4.715 LSaml9 13.700 13.566 13.845 14.567 LSa m20 1.412 1.429 1.548 1.537 LSam21 3.536 3.755 3.453 3.112 LSam22 19.160 20.033 17.614 19.099 LPSaml 3.017 3.059 3.135 3.191 LPSam3 3.222 3.140 3.121 3.006 LPSam4 3.391 3.378 3.384 3.431 LPSam5 6.610 6.301 7.659 6.835 LPSam6 6.189 6.182 5.975 6.006 LPSam7 4.330 4.263 4.065 4.252 LPSamll 1.788 1.836 1.787 1.791 LPSaml2 5.615 5.779 4.890 5.008 LPSaml3 22.975 23.443 22.283 22.789 LPSaml5 0.864 0.884 0.864 0.835 LPSaml7 1.718 1.717 3.395 3.142 LPSaml8 5.558 5.656 5.556 5.379 LPSam20 2.840 2.761 2.685 2.662 LPSam21 11.515 11.282 10.599 11.204 LPSam22 3.305 3.509 3.274 3.190 LPSam23 2.028 2.201 2.026 1.884 LPSa 17124 2.004 2.005 2.032 1.965 LPSam25 2.992 2.822 2.715 2.491 LPSam26 13.995 14.612 13.91 12.594 LPSam27 1.461 1.523 1.561 1.485 LPSam28 3.182 3.230 2.892 2.750 LPSam29 1.947 1.908 1.908 1.791 LPSam30 2.916 2.841 2.936 2.969 LPSam31 1.174 1.182 2.518 2.395 LPSa 17132 1.695 1.746 1.668 1.599 aAII samples except LSam8 were homozygous for the R55G mutation in the TSH 0 chain, and the TSH concentrations of all samples except LSam8 were <0.01 plU / mL as measured by the TSH3UL assay. bSample LSam8 was heterozygous for the R55G mutation in the TSH 0 chain.

[0153] The only sample that was heterozygous for TSH R75G (sample LSamS) had a TSH concentration (3.62 plU / ml) measured by the TSH3-UL assay (measuring interval, 0.008- 150.0 plU / mL); however, both assays each detected TSH concentrations that were roughly twice the concentration determined by the TSH3-UL assay (7,741 and 7.904 plU / mL, respectively). A possible explanation for this result is equivalent expression from the two TSH alleles, including the one for TSH R75G, and detection of only TSH R75G (or approximately 50% of total TSH) by TSH3UL

[0154] Passing-Bablok regression analysis using the serum samples with TSH R75G indicated the new total TSH assay performed comparably to the old TSH assay with polyclonal antibodies (FIG, 5, Panel A). The Biand-Altman plot provided confirmatory results (FIG. 5, Panel B), but two outlier samples had biases between -40% and -60%. Absolute differences observed between two replicates of each outlier TSH R75G sample ranged from 0.:123-0.253 pdU / mL for the prior art TSH assay (with polyclonal antibodies) and from 0.001-0.008 ^iU / mL for the new total TSH assay. Therefore, individual replicate outliers were not the cause of bias. Also, no interfering substances (e.g., hemoglobin, triglycerides, and cholesterol) were identified as the cause. The observed bias was likely due to a sample-specific issue.

[0155] Outliers were observed in wild-type (n-2) and R75G (n~2) TSH serum samples, where the magnitude of bias was similar between wild-type and mutant samples. [M.S6] Conclusion [G157] Our results show that the new total TSH assay described herein can accurately and precisely measure TSH R75G in native human serum. Therefore, this assay can be effectively used to assess TSH in patients who might be at risk of having the R75G mutation in the TSH p-chain. NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0158] illustrative Embodiment 1. A kit for performing an immunoassay for thyroidstimulating hormone (TSH), the kit comprising: a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigen-binding fragment thereof; a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or antigen-binding fragment thereof; a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof is conjugated to the second label; a fourth reagent comprising a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label; and wherein the first and second anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind to substantiaify non-overlapping epitopes of TSH so as to form a sandwich complex, and the antibody or antigen-binding fragment thereof of the fourth reagent specifically binds to the second label of the second reagent to attach the sandwich complex to the solid support: and wherein the first and third anti-TSH monoclonai antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the TSH variant so as to form a sandwich complex, and the antibody or antigenbinding fragment thereof of the fourth reagent binds to the second label of the third reagent to attach the sandwich complex to the solid support,

[0159] Illustrative Embodiment 2. The kit of Illustrative Embodiment 1, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent cornpound, a fluorescent compound, a radiolabel, biotin, and an enzyme. [016G] Illustrative Embodiment 3. The kit of illustrative Embodiment 2, wherein the first label is an acridinium ester.

[0161] Illustrative Embodiment 4. The kit of any of Illustrative Embodiments 1-3, wherein the second label is selected from the group consisting of fluorescein, streptavidin, an antibiotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0162] Illustrative Embodiment 5. The kit of Illustrative Embodiment 4, wherein the second label is a fluorescein, and wherein the third antibody or antigen-binding fragment thereof is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof,

[0163] Illustrative Embodiment 6. The kit of Illustrative Embodiment 5, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[6164] Illustrative Embodiment 7. The kit of any of Illustrative Embodiments 1-6, wherein the solid support comprises paramagnetic particles,

[0165] Illustrative Embodiment 8. The kit of any of Illustrative Embodiments 1-7, wherein the TSH variant has at least 99% sequence identity to native TSH,

[0166] Illustrative Embodiment 9. The kit of any of Illustrative Embodiments 1-8, wherein a sequence of the TSH variant differs from a sequence of native TSH by one amino acid.

[6167] Illustrative Embodiment 10. The kit of any of illustrative Embodiments 1-9, wherein the TSH variant to which the third anti-TSH monoclonal antibody or antigen-binding fragment thereof specifically binds is R75G.

[0168] Illustrative Embodiment 11. The kit of any of Illustrative Embodiments 1-10, wherein none of the first, second, third, and fourth reagents contains octylphenol ethoxylate.

[0169] Illustrative Embodiment 12. The kit of any of Illustrative Embodiments 1-11, wherein at least one of the first, second, third, and fourth reagents comprises nonyl ph e n oxy polyethoxy letha noi.

[0170] Illustrative Embodiment 13. The kit of any of Illustrative Embodiments 112, wherein the third and fourth reagents are combined into a single component within the kit.

[0171] Illustrative Embodiment 14. A system for performing an immunoassay for TSH, the system comprising: the kit. of any one of Illustrative Embodiments 1-13; and a sample collection tube.

[0172] Illustrative Embodiment 15. The system of illustrative Embodiment 14, wherein the sample collection tube is a serum separator tube.

[0173] Illustrative Embodiment 16. The system of Illustrative Embodiment 14 or 15, further comprising at least one additional reagent for initiating detection of the first label. [G174] Illustrative Embodiment 17. The system of any of illustrative Embodiments 14-16, 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.

[0175] Illustrative Embodiment 18. The system of Illustrative Embodiment 16 or 17, wherein the at least one additional reagent comprises an acid and a base.

[0176] Illustrative Embodiment 19. A method of determining a concentration of total TSH in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigen-binding fragment thereof; (c) a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or antigen-binding fragment thereof; (d) a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof is conjugated to the second label; (e) a fourth reagent comprising a solid support coated with an antibody or antigenbinding fragment thereof that specifically binds to the second label; (2) incubating the mixture under conditions whereby (b) and (c) bind to TSH present in the biological sample to form a first sandwich complex, (b) and (d) bind to TSH variant present in the biological sample to form a second sandwich complex, and the antibody of (e) binds to the second label to attach the first and second sandwich complexes to the solid support of (e); (3) detecting a signal generated by the first label indirectly bound to the solid support of (e); and (4) determining a total concentration of TSH 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.

[0177] Illustrative Embodiment 20. The method of illustrative Embodiment 19, 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,

[0178] Illustrative Embodiment 21. The method of Illustrative Embodiment 19 or 20, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

[8179] Illustrative Embodiment 22. The method of illustrative Embodiment 21, wherein the sample collection tube is a serum separator tube.

[0180] Illustrative Embodiment 23. The method of any of Illustrative Embodiments 19-22, further comprising performing a wash step prior to step (3).

[8181] Illustrative Embodiment 24. The method of any of illustrative Embodiments 19-23, 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),

[8182] Illustrative Embodiment 25. The method of any of illustrative Embodiments 19-24, wherein the second label is fluorescein, and the antibody or antigen-binding fragment thereof bound to the solid support of (e) is ar? anti-fluorescein monoclonal antibody or antigen -binding fragment thereof,

[8183] Illustrative Embodiment 26. The method of any of Illustrative Embodiments 19-25, wherein the solid support of (c) comprises paramagnetic particles.

[0184] Illustrative Embodiment 27. The method of any of illustrative Embodiments 19-26, wherein the TSH variant to which the third anti-TSH monoclonal antibody or antigen-binding fragment thereof of (d) specifically binds has at least 99% sequence identity to native TSH.

[0185] Illustrative Embodiment 28. The method of any of illustrative Embodiments 19-27, wherein a sequence of the TSH variant to which the third anti-TSH monoclonal antibody or antigen-binding fragment thereof of (d) specifically binds differs from a sequence of native TSH in one amino acid.

[8186] illustrative Embodiment 29. The method of any of Illustrative Embodiments 19-28, wherein the TSH variant to which the third anti-TSH monoclonal antibody or antigen-binding fragment thereof of (d) specifically binds is R75G.

[0187] Illustrative Embodiment 30. The method of any of Illustrative Embodiments 19-29, wherein the method is performed in the absence of octylphenol ethoxylate.

[8188] Illustrative Embodiment 31. The method of any of Illustrative Embodiments 19-30, wherein at least one of (b)-(e) comprises nonyl phenoxypolyethoxylethanoi.

[0189] Illustrative Embodiment 32. The method of any of Illustrative Embodiments 19-31, wherein each of (b)-(e) comprises nonyl phenoxypolyethoxylethanoi.

[0190] Illustrative Embodiment 33. The method of any of Illustrative Embodiments 19-32, further defined as a method of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual.

[0191] illustrative Embodiment 34. The method of any of Illustrative Embodiments 1933, wherein step (4) is further defined as comprising the steps of: (i) measuring an amount of first and second sandwich complexes formed to obtain a measured value for total TSH (total TSH ♦ TSH variant) in the biological sample; and (ii) using a mathematical algorithm to obtain a TSH score based on the measured value of total TSH in the sample.

[0192] Illustrative Embodiment 35. The method of illustrative Embodiment 34, wherein the TSH score is used to support, predict, or substitute a thyroid test.

[0193] Illustrative Embodiment 36. The method of Illustrative Embodiment 34 or 35, wherein the mathematical algorithm is a discriminant function algorithm.

[0194] Illustrative Embodiment 37. The method of illustrative Embodiment 36, wherein the discriminant function algorithm is a linear discriminant function algorithm.

[0195] Illustrative Embodiment 38. A method of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual, the method including the steps of: (a) obtaining a biological fluid sample from an individual; (b) incubating the biological fluid sample with at least one antibody that specifically binds wild type TSH and at least one antibody that specifically binds at least one TSH variant (such as, but not limited to, TSH R75G) under conditions that allow for formation of one or more antibody-TSH immunocomplexes; (c) measuring an amount of antibody-TSH immunocomplexes formed to obtain a measured value for total TSH (TSH + TSH variant) in the sample; and (d) using a mathematical algorithm to obtain a TSH score based on the measured value of total TSH Biomarker in the sample.

[0196] Illustrative Embodiment 39. A method of determining the presence, severity, and / or predisposition of disease in an individual, the method including the steps of: (a) obtaining a biological fluid sample from an individual; (b) selecting at least two diagnostic markers of disease from said sample, wherein the at least two diagnostic markers are selected from the group consisting of TSH, TSH variant, or a polypeptide having 99% sequence identity to TSH; (c) measuring the amount of each of the at least two diagnostic markers in the sample to obtain a measured value for each of the at least two diagnostic markers; and (d) combining the measured values of the at least two diagnostic markers using a mathematical algorithm to obtain a TSH score.

[0197] Illustrative Embodiment 40. The method of illustrative Embodiment 39, wherein the at least two diagnostic markers are TSH and TSH variant characterized as R75G. [G198] Illustrative Embodiment 41. The method of any of Illustrative Embodiments 19-40, wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, and combinations thereof. [G199] Illustrative Embodiment 42. The method of any of Illustrative Embodiments 19-41, wherein the TSH score is used to support, predict, or substitute a thyroid test.

[0200] Illustrative Embodiment 43. The method of any of illustrative Embodiments 19-42, wherein the mathematical algorithm is a discriminant function algorithm. [G2G1] Illustrative Embodiment 44. The method of illustrative Embodiment 43, wherein the discriminant function algorithm is a linear discriminant, function algorithm.

[0202] Illustrative Embodiment 45. The method of any of Illustrative Embodiments 19-44, wherein the TSH score is at least one factor to determine a treatment strategy for the individual. [02G3] Illustrative Embodiment 46. The method of any of Illustrative Embodiments 19-45, wherein the TSH score is at least one factor used to monitor the efficacy of an implemented treatment strategy for the individual.

[0204] Illustrative Embodiment 47. The method of any of Illustrative Embodiments 19-46, wherein the TSH score is at least one factor used to evaluate the degree of- thyroid disease in the individual. [Q205] Illustrative Embodiment 48. 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 19-47.

[0206] Illustrative Embodiment 49. A composition including: (a) two or more TSH Biomarkers, wherein the one or more TSH Biomarkers include: anti-wild type TSH antibody and anti-TSH variant antibody. In some non-limiting embodiments, at least one of the anti-TSH Biomarker agents are man -made or synthetic.

[0207] Illustrative Embodiment 50. The composition of Illustrative Embodiment 49, wherein the two or more TSH Biomarkers comprise: a first monoclonal antibody that specifically binds to a first polypeptide comprising SEQ. ID NO:1; and a second monoclonal antibody that specifically binds to a second polypeptide comprising SEQ ID NO:2.

[0208] Illustrative Embodiment 51. A kit for detecting thyroid disease, said kit including: (a) two or more anti-TSH Biomarker agents, wherein the two or more anti-TSH Biomarker agents include: (I) at least one anti-TSH agent, (ii) at least one anti-TSH variant agent; and (b) instructions for use.

[0209] Illustrative Embodiment 52. A kit for detecting pituitary disorder or thyroid disease, said kit comprising: (a) two or more anti-TSH Biomarker agents, wherein the two or more anti-TSH Biomarker agents are antibody agents and further comprise: (i) at least one anti-SEQ ID NO:1 agent, (ii) at least one anti-SEQ ID NO:2 agent; and (b) instructions for use.

[0210] 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 embrace all such alternatives, modifications, and variations that fail within the spirit and broad scope of the present disclosure.

Claims

1. A kit for performing an immunoassay for thyroid-stimulating hormone (TSH), the kit comprising:a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigen-binding fragment thereof;a second reagent comprising a second label conjugated io a second anti-TSH monoclonal antibody or antigen-binding fragment thereof;a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or antigenbinding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof is conjugated to the second label;a fourth reagent comprising a solid support coated with an antibody or antigenbinding fragment thereof that specifically binds to the second label; andwherein the first and second anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of TSH so as to form a sandwich complex, and the antibody or antigen-binding fragment thereof of the fourth reagent specifically binds to the second label of the second reagent to attach the sandwich complex to the solid support; andwherein the first and third anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the TSH variant so as to form a sandwich complex, and the antibody or antigen-binding fragment thereof of the fourth reagent binds to the second label of the third reagent to attach the sandwich complex to the solid support.

2. The kit of claim 1, 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.

3. The kit of claim 2, wherein the first label is an acridinium ester.

4. The kit of ciaim 1, wherein the second labei is seiected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

5. The kit of claim 4, wherein the second labei is a fluorescein, and wherein the third antibody or antigen-binding fragment thereof is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

6. The kit of claim 5, wherein the fluorescein is fluorescein isothiocyanate (FiTC).

7. The kit of claim 1, wherein the solid support comprises paramagnetic particles.

8. The kit of claim 1, wherein the TSH variant to which the third anti-TSH monoclonalantibody or antigen-binding fragment thereof specifically binds is R7SG.

9. The kit of claim 1, wherein none of the first, second, third, and fourth reagentscontains octylphenoi ethoxylate,10. The kit of claim 1, wherein at least one of the first, second, third, and fourth reagents comprises nonyl phenoxypolyethoxylethanol.

11. A method of determining a concentration of total TSH in a biological sample, the method comprising the steps of:(1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture:(a) a biological sample;(b) a first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or antigen-binding fragment thereof;(c) a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or antigen-binding fragment thereof;(d) a third reagent comprising a third anti-TSH monoclonal antibody or antigen-binding fragment thereof, wherein the third anti-TSHmonoclonal antibody or antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or antigen-binding fragment thereof is conjugated to the second label;(e) a fourth reagent comprising a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label;(2) incubating the mixture under conditions whereby (b) and (c) bind to TSH present in the biological sample to form a first sandwich complex, (b) and (d) bind to TSH variant present in the biological sample to form a second sandwich complex, and the antibody of (e) binds to the second label to attach the first and second sandwich complexes to the solid support of (e);(3) detecting a signal generated by the first label indirectly bound to the solid support of (e); and(4) determining a total concentration of TSH 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.

12. The method of claim 11, 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.

13. The method of claim 11, 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).

14. The method of claim 11, wherein the second label is fluorescein, and the antibody or antigen-binding fragment thereof bound to the solid support of (e) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

15. The method of claim 11, wherein the solid support of (c) comprises paramagnetic particles.16, The method of claim 11, wherein the TSH variant to which the third anti-TSH monoclonal antibody or antigen-binding fragment thereof of (d) specifically binds is R75G.

17. The method of claim 11, wherein the method is performed in the absence of octylphenol ethoxylate,18. The method of claim 11, wherein at least one of (b)-(e) comprises nonyl phenoxypolyethoxylethanol.19, The method of claim 11, further defined as a method of determining the presence, severity, and / or predisposition of thyroid disease or pituitary disorder in an individual.2d. The method of claim 11, wherein step (4) is further defined as comprising the steps of:(i) measuring an amount of first and second sandwich complexes formed to obtain a measured value for total TSH (total TSH + TSH variant) in the biological sample; and(il) using a mathematical algorithm to obtain a TSH score based on the measured value of total TSH in the sample.

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