Sandwich assays for tsh biomarker detection and methods of production and use thereof

Abstract

Disclosed are compositions, kits, systems, and methods for determining total TSH concentration in a biological sample and / or determining the presence, severity, and / or susceptibility of a thyroid disease and / or pituitary disorder in an individual. 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, such that at least an antibody-TSH-antibody sandwich immunocomplex and an antibody-TSH variant-antibody sandwich immunocomplex can be formed. The methods include determining the amount of total TSH present in the biological sample based on the amounts of the two sandwich immunocomplexes formed.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 562,449, filed March 7, 2024. The entire contents of the aforementioned patent application are expressly incorporated herein by reference.

[0002] References to the electronically submitted sequence list This application contains a sequence list that is a separate part of this disclosure, submitted via EFS-Web in computer-readable form as an XML file. The sequence list, created on February 24, 2025, is named “202404459_Sequence_Listing” and has a size of 4,803 bytes. The entire contents of the sequence list are incorporated herein by reference. Background Technology

[0003] Some immunoassays detect a complex between a target biomarker, a first biomarker-binding antibody, and a labeled second biomarker-binding antibody. In one example of a sandwich immunoassay, the first biomarker-binding antibody, such as a capture antibody suitable for binding the target biomarker, is attached to an insoluble material or matrix to form a solid-phase reagent. A luminescent reagent comprising a second antibody, which also binds the target biomarker at an epitope that substantially does not overlap with the epitope bound by the first antibody, is also provided; the second antibody, for example, includes a labeled antibody comprising a signal portion. When contacted under predetermined conditions with a biological sample comprising a predetermined antigen or target biomarker, the solid-phase reagent, the target biomarker, and the luminescent reagent form a complex capable of generating a detectable signal indicating the presence and / or amount of the target biomarker.

[0004] Biomarkers serve as surrogate indicators of clinically meaningful outcomes and may or may not reflect the underlying pathogenesis of a disease. Examples of clinical utility include diagnosis, prediction of disease progression or regression, and prognosis of mortality. Biomarkers should be readily available, reliably measurable, and usable for continuous monitoring. Ideally, biomarkers also offer advantages in terms of convenience, timeliness, and / or cost compared to currently used clinical measurements.

[0005] However, although assays for detecting biomarkers are available, variants of biomarkers that may be less easily or accurately measured have been identified in certain immunoassays. For example, the known biomarker thyroid-stimulating hormone (TSH) can be present in biological samples in the form of mutant variants with mutations in the β subunit, causing problems with reduced detectability in some immunoassays. For example, Shaki et al. ( Eur Thyroid J.,2022, 11(1)e210072) explained that the homozygosity of the TSHB p.R75G variant caused some immune detection-based platforms to fail TSH tests, leading to misdiagnosis of hyperthyroidism.

[0006] Therefore, there is a need in the art for novel and improved immunoassays for the detection of target biomarkers, wherein the target biomarkers may include one or more isotypes, variants, or alterations that may reduce the power and / or accuracy of the assay. This disclosure relates precisely to such biomarkers, and compositions / kits / systems / devices / assays containing reagents for measuring such biomarkers, together with methods of use thereof. Brief description of the attached diagram The embodiments of this disclosure, which have been briefly summarized above and discussed in more detail below, can be understood by referring to the illustrative embodiments of this disclosure depicted in the accompanying drawings. However, the drawings only show typical embodiments of this disclosure and should therefore not be considered as limiting the scope, as other equally effective embodiments of this disclosure are possible.

[0008] Figure 1 A non-limiting embodiment of an immunoassay is described, the immunoassay being constructed according to the present disclosure and utilizing a first capture monoclonal antibody (mAb) and a second capture mAb in a solid-phase reagent to prepare a semi-prepared solid phase suitable for sandwich immunoassays.

[0009] Figure 2 An example block diagram of a non-limiting embodiment of a computer system 1100 configured for use in accordance with this disclosure is depicted.

[0010] Figure 3 An exemplary flowchart of a non-limiting embodiment of method 1200 according to this disclosure is depicted.

[0011] Figure 4 An exemplary flowchart of another non-limiting embodiment of method 1300 according to this disclosure is depicted.

[0012] Figure 5 Passing-Bablok regression (A) and Bland-Altman plot (B) of the sample, which was confirmed to have a TSH R75G mutation, are graphically depicted using existing TSH2 and recently developed total TSH assays in ADVIA CENTAUR. ®The analysis was performed on the XP system (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY). In small plot A, the upper line is the Passing-Bablok regression line; the lower line is the line of identity.

[0013] For ease of understanding, the same reference numerals have been used to denote common elements in the accompanying drawings, where possible. The drawings are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated into other embodiments without further description. Summary of the Invention

[0014] Before explaining in detail at least one embodiment of this disclosure through exemplary language and results, it should be understood that this disclosure is not limited in its application to the construction details and component arrangements set forth in the description below. This disclosure can have other embodiments, or can be practiced or carried out in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning; and the embodiments are intended to be exemplary, not exhaustive. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0015] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and described in the various general and more specific references cited and discussed throughout this specification. The nomenclature, laboratory procedures, and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art.

[0016] All patents, published patent applications, and non-patent publications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the extent that each individual patent or publication specifically and individually indicates its inclusion by reference.

[0017] Based on this disclosure, all compositions, devices, kits, and / or methods disclosed herein can be prepared and performed without excessive experimentation. While compositions, devices, kits, and / or methods have been described according to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions, devices, kits, and / or methods described herein, as well as the steps or sequence of steps of the methods, without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0018] Unless otherwise stated, the following terms should be understood to have the following meanings when used in accordance with this disclosure: When used in conjunction with the term "comprising" in the claims and / or specification, the term "a" or "an" may mean "one / a," but it is also consistent with the meaning of "one or more / a combination of," "at least one / a," and "one / a or more than one / a." In this way, unless the context clearly indicates otherwise, the terms "a," "an," and "the / described" include plural indicators. Therefore, for example, reference to "compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "a plurality" means "two or more."

[0019] The term "at least one" should be understood to include any quantity of one or more, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term it is attached to; furthermore, a quantity of 100 / 1000 is not considered limiting, as higher limits can also produce satisfactory results. Additionally, the term "at least one of X, Y, and Z" should be understood to include a single X, a single Y, and a single Z, as well as any combination of X, Y, and Z. Similarly, the term "at least two" should be understood to include any quantity of two or more, including but not limited to 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.

[0020] For example, the use of ordinal terms (i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of distinguishing two or more items and, unless otherwise expressly stated, is not intended to imply any order or sequence, or that one item is more important than another, or any additional order.

[0021] The use of the term "or" in the claims is intended to mean inclusive "and / or" unless it is explicitly stated that it refers only to substitutes or unless the substitutes are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0022] 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 an embodiment is included in at least one embodiment. For example, the phrases “some embodiments” or “one example” appearing throughout the specification do not necessarily refer to the same embodiment. Furthermore, all references to one or more embodiments or examples are construed as non-limiting to the claims.

[0023] Throughout this application, the term "about" is used to indicate values ​​including inherent error variations in the composition / instrument / device, variations in the method used to determine the value, or variations present in the subject of study. For example, but not as a limitation, when using the term "about," a specified value can vary from plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, as such variation is suitable for performing the disclosed method and as understood by one of ordinary skill in the art.

[0024] The term "antibody" is used herein in its broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and their conjugates (e.g., but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, biantibodies, single-chain antibodies, and other antibody fragments and their conjugates that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., modified binding proteins / peptides), and combinations or derivatives thereof. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0025] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain bound together in a tight, non-covalent manner. Extending from the folds of these two domains are six hypervariable rings (three from the H chain and three from the L chain), which facilitate the amino acid residues used for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three complementarity-determining regions (CDRs) for antigen specificity) has the ability to recognize and bind antigens, albeit with an affinity lower than that of the entire binding site. As used herein, "antibody heavy chain" refers to the larger of the two classes of polypeptide chains present in their native conformation in all antibody molecules. As used herein, "antibody light chain" refers to the smaller of the two classes of polypeptide chains present in their native conformation in all antibody molecules. K and A light chains refer to the two major antibody light chain isotypes.

[0026] As used herein, the term "synthetic antibody" means an antibody generated using recombinant DNA technology, such as antibodies expressed by bacterial phages as described herein. The term should also be interpreted as meaning an antibody generated by synthesizing a DNA molecule encoding an antibody, and said DNA molecule expressing an antibody protein or an amino acid sequence specifying an antibody, wherein said DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0027] As used herein, the term "antigen" is defined as a molecule that elicits an immune response, which may involve antibody production, activation of specific immune-active cells, or both. Antigens can include any macromolecule, including virtually all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA comprising a nucleotide sequence or a portion thereof (encoding a protein that elicits an immune response) thus encodes an "antigen" as used herein. Furthermore, an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Further, antigens can be generated, synthesized, or derived from biological samples, including tissue samples, tumor samples, cells, or biological fluids.

[0028] The term "immunoglobulin" or "Ig" refers to a class of proteins that act as antibodies. Five members of this class are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody found in bodily fluid secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that does not have a known antibody function but can act as an antigen receptor. IgE is such an immunoglobulin that, upon exposure to an allergen, mediates an immediate hypersensitivity reaction by inducing the release of mediators from mast cells and basophils.

[0029] "Separated" means material that is substantially or substantially free of the components that normally accompany it in its natural state. For example, as used herein, "separated polynucleotide" refers to a polynucleotide that has been purified from a sequence that is side-linked to it in its natural state, such as a DNA fragment that has been removed from a sequence that is normally adjacent to the fragment. Alternatively, as used herein, "separated peptide" or "separated polypeptide," etc., refers to peptide or polypeptide molecules that have been isolated and / or purified in vitro from their natural cellular environment and from their binding with other components of the cell.

[0030] In the context of this disclosure, the following abbreviations are used for commonly used nucleic acid bases: “A” for adenosine, “C” for cytosine, “G” for guanosine, “T” for thymidine, and “U” for uridine.

[0031] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Phrases encoding protein or RNA nucleotide sequences may also include introns, to the extent that protein-encoding nucleotide sequences may contain introns in some forms.

[0032] The terms “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (e.g., chemical analogs of the corresponding naturally occurring amino acids), as well as naturally occurring amino acid polymers. In some respects, polypeptides may include enzymatic polypeptides or “enzymes,” which typically catalyze (i.e., increase their rate) various chemical reactions.

[0033] The term "variant" refers to a polypeptide that differs from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In some non-limiting embodiments, the polypeptide variant differs from the reference polypeptide by one or more substitutions, which may be conserved or non-conserved. In some non-limiting embodiments, the polypeptide variant contains conserved substitutions (e.g., 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 other amino acids having broadly similar properties without altering the polypeptide's activity. The term "polypeptide variant" also encompasses polypeptides in which one or more amino acids have been added, deleted, or substituted with different amino acid residues. In some non-limiting embodiments, the term refers to a variant as described in U.S. Patent Application Publication No. 2018 / 0222995.

[0034] Consistent with its use in the art, the term "biomarker" or "biological marker" is used herein to refer to an entity whose presence, level, or form is associated with a particular target biological event or state, such that it is considered a "marker" of that event or state. To name just a few examples, in some non-limiting embodiments, a biomarker may be or include markers about a particular disease state, or the likelihood that a particular disease, symptom, or condition may develop, occur, or recur. In some non-limiting embodiments, a biomarker may be or include markers about a particular disease or treatment outcome or its likelihood. Thus, in some non-limiting embodiments, a biomarker is a prediction, prognosis, and / or diagnosis of a relevant target biological event or state. In some non-limiting embodiments, a biomarker is a possible biomarker of a relevant target biological event or state. A biomarker can be an entity of any chemical class. For example, in some non-limiting embodiments, a biomarker may be or include nucleic acids, peptides, small molecules, or combinations 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, the biomarker is found in a specific tissue (e.g., lung tissue). In some non-limiting embodiments, the biomarker is found extracellularly (e.g., secreted or otherwise generated or present outside the cells, such as in bodily fluids like blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0035] As described herein, in some non-limiting embodiments, the biomarker is a thyroid-stimulating hormone (TSH) or TSH biomarker. As used herein, “TSH biomarker” refers to a biomarker relating to thyroid or pituitary disorders. In some non-limiting embodiments, one or more TSH biomarkers include a TSHB p.R75G variant (based on the utilized Human Genome Assembly, also referred to as “R55G,” as described in detail in Example 2) or a combination thereof. In some non-limiting embodiments, a TSH biomarker includes a gene product associated with a specific said biomarker. For example, depending on the context, “TSH” refers to a nucleotide encoding TSH or a characteristic or functional fragment thereof, and a TSH protein or a characteristic or functional fragment thereof. In some non-limiting embodiments, a TSH biomarker includes a variant of TSH comprising a protein having an amino acid sequence having at least 90%, 95%, or 99% sequence identity with wild-type TSH or its β subunit (SEQ ID NO: 1). In some non-limiting embodiments, the TSH biomarker includes a TSH hormone in which its β subunit has a modified substitution, for example, at position 75 (R75G; SEQ ID NO:2) used for numbering in SEQ ID NO:1.

[0036] The term "characteristic fragment" refers to a fragment of a biomarker (e.g., a TSH biomarker) that is sufficient to identify biomarkers derived from the fragment. For example, in some non-limiting embodiments, a "characteristic fragment" of a biomarker is a fragment containing an amino acid sequence or set of amino acid sequences that collectively allow a biomarker derived from the fragment to be distinguished from other possible biomarkers, proteins, or peptides. In some non-limiting embodiments, the characteristic fragment comprises at least 10, at least 20, at least 30, at least 40, or at least 50 amino acids. In some 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 with a biomarker derived from the characteristic fragment. In some non-limiting embodiments, SEQ ID NO:1 is a characteristic fragment of a TSH biomarker.

[0037] As used herein, the term “gene product” generally refers to RNA transcribed from a gene (before and / or after processing) or a polypeptide encoded by RNA transcribed from that gene (before and / or after modification).

[0038] As used herein, the term "detection reagent" refers to any detectable element, molecule, functional group, compound, fragment, or part. In some non-limiting embodiments, the detection reagent is provided or used alone. In some non-limiting embodiments, the detection reagent is provided and / or used in combination (e.g., linking) with another reagent. Examples of detection reagents include, but are not limited to: various ligands, radionuclides (e.g., 3 H, 14 C 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu、 187 Re、 111 In、 90 Y、 99m Tc, 177 Lu、 89 Zr, fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stable dioxanes, etc.), bioluminescent agents, spectrally resolved inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins relating to which antiserum or monoclonal antibodies can be obtained. In some non-limiting embodiments, suitable detection reagents for use herein include acridinium esters such as those described in U.S. Patent No. 6,664,043. In some non-limiting embodiments, suitable acridinium esters include HEGAE as described in U.S. Patent No. 6,664,043.

[0039] As used herein, a “diagnostic test” is a step or series of steps performed or already performed to obtain information that can be used to determine whether a patient has a disease, symptom, or condition and / or to classify the disease, symptom, or condition into a phenotypic category or any category that has prognostic significance regarding the disease, symptom, or condition, or is likely to respond to treatment (general treatment or any specific treatment) for the disease, symptom, or condition. Similarly, “diagnosis” refers to providing any type of diagnostic information, including but not limited to whether a subject is likely to have or develop a disease, symptom, or condition, such as the state, stage, or characteristics of the disease, symptom, or condition exhibited in the subject, information related to the nature or classification of a tumor, information related to prognosis, and / or information that can be used to select appropriate treatment or additional diagnostic tests. Treatment selection may include the selection of a specific therapeutic agent or other treatment modality such as surgery, radiation, etc., the choice of whether to discontinue or continue the therapy, choices related to the dosing regimen (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents), etc. The selection of additional diagnostic tests may include tests that are more specific to a given disease, symptom, or condition.

[0040] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “contain” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional elements or method steps not listed. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements but may include other elements not expressly listed or inherently present therein.

[0041] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is significant in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so on. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limitation on the number of items or terms in any combination.

[0042] As used herein, the term "substantially" means that the event or situation subsequently described occurs completely, or occurs to a large extent or to a large degree. For example, when relating to a particular event or situation, the term "substantially" means that the event or situation subsequently described occurs at least 80% of the time, or at least 85% of the time, at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within close proximity to each other but not 100% adjacent, or that a portion of one item is not 100% adjacent to the other item but is within close proximity to the other item.

[0043] As used herein, the phrase “related to” includes both direct and indirect relationships between two parts. Non-limiting examples of relatedness include covalent bonding of one part to another via a direct bond or via a spacer group; non-covalent bonding of one part to another directly or by means of a specific binding pair member of the part; and incorporation of one part into another, for example, by dissolving one part into another or by synthesis and coating one part onto another.

[0044] As used herein, the term "biofluid sample" should be understood to include any liquid test sample that can be obtained from a patient and utilized in accordance with this disclosure. Examples of biofluid samples that can be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, mucus, nose, nasopharynx, anterior nose, oropharynx, trachea, bronchoalveolar fossa, cerebrospinal fluid (CSF), intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, tears, combinations thereof, etc.

[0045] As used herein, when the term “volume” refers to a liquid test sample utilized in accordance with this disclosure, it generally refers to the volume of a liquid test sample ranging from about 0.1 µl to about 100 µl, or from about 1 µl to about 75 µl, or from about 2 µl to about 60 µl, or less than or equal to about 50 µl.

[0046] As used herein, the term “Triton X-100” refers to a nonionic surfactant having a hydrophilic polyethylene oxide chain and a lipophilic or hydrophobic aromatic hydrocarbon group. Triton X-100 can be referred to by the following chemical names: “octylphenol polyoxyethylene ether”, “polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether”, “tert-octylphenoxypolyethoxyethanol”, and “polyethylene glycol tert-octylphenyl ether”.

[0047] As used herein, the term “TERGITOL™ 15-S-9” (Dow Chemical Company) refers to it as a nonionic surfactant derived from secondary alcohol ethoxylates. TERGITOL™ 15-S-9 can also be referred to by the name NP-40 and the chemical name “nonylphenol polyoxyethylene ether”.

[0048] As used herein, the term "patient" includes both humans and veterinary subjects. In some non-limiting embodiments, the patient is a mammal. In some other non-limiting embodiments, the patient is a human. For diagnostic / therapeutic purposes, the term "mammal" means any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo animals, sporting animals, or pet animals such as dogs, horses, cats, cattle, etc.

[0049] "Healthcare provider" or "healthcare 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 for treating lung diseases, a healthcare provider may be an individual not authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient.

[0050] As used herein specifically (but not as a limitation), the term "specific binding coupler" should be understood to mean any molecule capable of specifically binding to a target analyte or target biomarker. For example, but not as a limitation, a binding coupler can be an antibody, receptor, ligand, aptamer, molecularly imprinted polymer (i.e., an inorganic matrix), combinations or derivatives thereof, and any other molecule capable of specifically binding to a target analyte.

[0051] As used herein, the term "immunoassay" refers to an assay that determines the presence of a diagnostic biomarker in a biological sample by reacting the sample with an antibody (or a fragment thereof) that specifically binds to the diagnostic biomarker, wherein the reaction is performed under conditions and at a time that allow for the formation of an immune complex between the antibody (or a fragment thereof) and the diagnostic biomarker. In some non-limiting embodiments, quantification of such immune complexes is then performed. In some specific (but non-limiting) embodiments, the immunoassay may use a labeled second antibody that binds to a first antibody to detect an immobilized complex between a serum marker and a serum marker-binding antibody. Alternatively, the first form is characterized as a sandwich assay in which the second antibody also binds to the serum marker. In a sandwich immunoassay procedure, the serum marker-binding antibody may be a capture antibody attached to an insoluble material, while the second antibody may be a labeled antibody. The sandwich immunoassay procedure described above may be used in conjunction with the antibodies described below.

[0052] In other specific (but not limiting) embodiments, the immunoassay may use a detection molecule (i.e., a second reagent) to detect a complex between a serum marker and a serum marker-binding antibody, said detection molecule being capable of binding to the serum marker-binding antibody and also being detectable upon binding to the immune complex. For example (but not as a limitation), the second reagent may include a marker attached to a receptor, a ligand, or even another copy of the serum marker.

[0053] Turning now to specific non-limiting embodiments of this disclosure, embodiments of which include various biomarkers for TSH, TSH homologs, isotypes, and variants, and compositions / kits / systems / devices / assays containing them, methods of their manufacture and use, and diagnostic tests associated therewith. The identification of one or more biomarkers for TSH is attractive and advantageous for several reasons. First, currently used diagnostic criteria for TSH can be limited by the failure to detect one or more variants of TSH. The identification of one or more diagnostic serum biomarkers for TSH is helpful to both clinicians and patients, particularly where the genetic background of the individual or population to be tested produces variants of TSH that are difficult or impossible to detect in current assays.

[0054] This disclosure relates to biomarkers, and compositions / reagents / kits / systems / devices / assays including reagents for measuring such biomarkers, together with methods of using them. In some non-limiting embodiments, the biomarker is wild-type TSH and a variant of TSH.

[0055] In some non-limiting embodiments, this disclosure includes compositions, reagents, kits, systems, and methods for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorders in an individual. The method includes the steps of: (a) obtaining a biofluid sample from the individual; (b) incubating the biofluid sample with at least one antibody specifically binding to TSH and at least one antibody binding to a TSH variant (e.g., but not limited to TSH R75G) under conditions allowing the formation of at least two antibody-TSH immune complexes (i.e., one immune complex containing wild-type TSH and another containing a TSH variant); (c) measuring the amount of the formed antibody-TSH immune complexes to obtain a measurement of total TSH (wild-type TSH + TSH variant) in the sample; and (d) using a mathematical algorithm to obtain a TSH score based on the measurement of total TSH biomarkers in the sample.

[0056] In some non-limiting embodiments, this disclosure includes compositions, reagents, kits, systems, and methods for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorders in an individual. The method includes the steps of: (a) contacting a biofluid sample from an individual with at least one antibody specifically binding wild-type TSH and at least one antibody binding a TSH variant (e.g., but not limited to TSH R75G) under conditions allowing the formation of at least two antibody-TSH immune complexes (i.e., one immune complex containing wild-type TSH and another containing a TSH variant); (b) measuring the amount of the formed antibody-TSH immune complexes to obtain a measurement of total TSH (wild-type TSH + TSH variant) in the sample; and (c) using a mathematical algorithm to obtain a TSH score based on the measurement of total TSH biomarkers in the sample.

[0057] In certain specific (but non-limiting) embodiments, at least two diagnostic markers include TSH and TSH variants, and step (d) is further defined as combining measurements of the two diagnostic markers using a mathematical algorithm to obtain a TSH score. In certain non-limiting embodiments, TSH and TSH variants each represent a person.

[0058] In some non-limiting embodiments, this disclosure includes compositions, reagents, kits, systems, and methods for determining the presence, severity, and / or susceptibility to a disease in an individual. The method includes the steps of: (a) obtaining a biological fluid sample from the individual; (b) selecting at least two diagnostic markers for the disease from the sample, wherein the at least two diagnostic markers are selected from TSH, TSH isotypes, TSH variants (e.g., but not limited to R55G / R75G), or peptides having 99% sequence identity with TSH, and combinations thereof; (c) measuring the amount of each of the at least two diagnostic markers in the sample to obtain a measurement value for each of the at least two diagnostic markers; and (d) combining the measurements of the at least two diagnostic markers using a mathematical algorithm to obtain a TSH score (e.g., but not limited to, a TSH score for thyroid or pituitary disease). In some non-limiting embodiments, the at least two diagnostic markers are TSH and a TSH variant characterized by R55G / R75G, wherein the TSH variant includes a substitution of R for G at position 55 / 75 (depending on the nomenclature of the human genome assembly utilized). In other non-limiting embodiments, at least two diagnostic markers are TSH and a TSH variant that has at least 99% sequence identity with wild-type TSH.

[0059] In some non-limiting embodiments, this disclosure includes a kit for detecting thyroid diseases, the kit comprising: (a) one or more anti-TSH biomarker reagents, wherein the one or more anti-TSH biomarker reagents comprise: (i) an anti-TSH reagent, (ii) an anti-TSH variant reagent; and (b) instructions for use. In some non-limiting embodiments, the one or more anti-TSH biomarker reagents are artificial or synthetic. In some non-limiting embodiments, two or more anti-TSH reagents comprise: a first antibody that specifically binds to a peptide comprising SEQ ID NO:1; and a second antibody that specifically binds to a peptide comprising SEQ ID NO:2.

[0060] In some non-limiting embodiments, this disclosure relates to a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform one or more operations comprising any of the embodiments described herein.

[0061] This disclosure also provides a kit comprising one or more anti-TSH biomarker reagents and instructions for use (e.g., for therapeutic, preventative, or diagnostic purposes), 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 disorders, or combinations thereof. In some non-limiting embodiments, one or more anti-TSH biomarker reagents include antibody reagents. In some non-limiting embodiments, two or more antibody reagents are labeled with a detectable portion. In some non-limiting embodiments, the kit further includes a detection reagent (e.g., one or more acridinium ester molecules). In some non-limiting embodiments, one or more antibody reagents are labeled with one or more acridinium ester molecules such as (but not limited to) HEGAE. In some non-limiting embodiments, the kit further includes one or more second antibody reagents that specifically bind to one or more anti-TSH biomarker antibody reagents.

[0062] In some non-limiting embodiments, the kit further includes one or more control samples. In some non-limiting embodiments, the control samples include one or more TSH biomarker standards.

[0063] In addition to the above, the kit may also include other components, such as solvents or buffers, stabilizers or preservatives, TERGITOL™ (Dow Chemical Company), and / or reagents for treating the conditions or symptoms described herein. Alternatively, other components may be included in the kit, but in a different composition or container than the anti-TSH biomarker reagent. In such embodiments, the kit may include instructions for mixing the anti-TSH biomarker reagent with other components, or for using the anti-TSH biomarker together with other components.

[0064] In some non-limiting embodiments, the kit used in accordance with this disclosure may include one or more other components, such as (but not limited to) a reference or control sample, instructions for processing the sample, instructions for performing a test on the sample, instructions for interpreting the results, buffer solutions, and / or other reagents required for performing the test.

[0065] This document also provides compositions. In some non-limiting embodiments, the composition comprises two or more of any anti-TSH biomarker reagents as described herein or otherwise considered. In some non-limiting embodiments, the two or more TSH biomarkers comprise anti-TSH biomarker reagents and anti-TSH variant biomarker reagents, or combinations thereof. In some non-limiting embodiments, the composition comprises, individually or collectively as a complex, […]. Figure 1 The composition in.

[0066] Certain non-limiting embodiments of this disclosure relate to a kit for performing an immunoassay for thyroid-stimulating hormone (TSH). The kit includes: a first reagent containing a first label conjugated to a first anti-TSH monoclonal antibody or an antigen-binding fragment thereof; a second reagent containing a second label conjugated to a second anti-TSH monoclonal antibody or an antigen-binding fragment thereof; a third reagent containing a third anti-TSH monoclonal antibody or an antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof is conjugated to the second label; and a fourth reagent containing a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label. First and second anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind substantially non-overlapping epitopes of TSH to form a sandwich complex, and an antibody of a fourth reagent or an antigen-binding fragment thereof specifically binds a second label of the second reagent to attach the sandwich complex to a solid support; and wherein the first and third anti-TSH monoclonal antibodies or antigen-binding fragments thereof bind substantially non-overlapping epitopes of TSH variants to form a sandwich complex, and the antibody of the fourth reagent or an antigen-binding fragment thereof binds a second label of the third reagent to attach the sandwich complex to a solid support.

[0067] Certain non-limiting embodiments of this disclosure relate to systems for performing immunoassays for TSH, said systems including any kits disclosed herein or otherwise considered. The system may further include one or more other components; for example, sample collection tubes (e.g., but not limited to serum separation tubes) and / or at least one additional reagent for initiating a first labeling detection.

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

[0069] In one particular (but not limiting) implementation, the method is further defined as a method for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorder in an individual.

[0070] In one particular (but not limiting) embodiment, step (4) of the method is further defined as including the following steps: (i) measuring the amount of the first sandwich complex and the second sandwich complex formed to obtain a measurement of total TSH (total TSH + TSH variant) in the biological sample; and (ii) using a mathematical algorithm (e.g., but not limited to a discriminant function algorithm, i.e., a linear discriminant function algorithm) to obtain a TSH score based on the measurement of total TSH in the sample. This TSH score can be used (e.g., but not limited to) to support, predict, or replace thyroid testing.

[0071] Any biofluid sample known in the art or otherwise contemplated herein may be used in accordance with this disclosure, which may contain TSH or TSH variants detectable by two or more anti-TSH antibodies and indicating thyroid disease or pituitary disorders. Examples of biofluid samples that may be used include, but are not limited to, blood, serum, plasma, saliva, sputum, mucus, nose, nasopharynx, anterior nose, oropharynx, trachea, bronchoalveolar fossa, combinations thereof, etc. In some non-limiting embodiments, the biofluid may be modified and / or may include human serum and plasma (EDTA and lithium heparin).

[0072] Any anti-TSH antibody known in the art or otherwise contemplated herein may be used in accordance with this disclosure. Anti-TSH antibodies are well known in the art and are commercially available. For example (but not as a limitation), anti-TSH antibodies that may be used as antibodies binding to a first label and a second label in accordance with this disclosure include those disclosed in: U.S. Patent Application Publication Nos. US2009 / 0087923; US2009 / 0088334; US2012 / 0301905; US2012 / 0301906; US2012 / 0301896; and US2016 / 0109466; and U.S. Patent Nos. 8,253,445; 8,617,826; 9,068,994; 9,199,234; and 9,575,077.In addition, anti-TSH monoclonal antibodies available according to this disclosure are available from a variety of commercial sources, including (but not limited to) AbboMax (San Jose, CA); Abcam (Cambridge, UK); Anogen (Mississauga, Ontario); Antibodies-Online Inc. (Limerick, PA); Aviva Systems Biology (San Diego, CA); BBI Solutions (Portland, ME); Biocare Medical (Concord, CA); Biobyt (Cambridge, UK); Bioss Inc. (Woburn, MA); BosterBio (Pleasanton, CA); CreativeBiolabs, 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, MA); US Biological LifeSciences (Salem, MA); and many other sources. However, this list is not exhaustive, and many other commercial sources exist for antibodies that can be utilized according to this disclosure. Therefore, those skilled in the art will be able to clearly and unambiguously identify and select various monoclonal antibodies that can be utilized according to this disclosure, and thus, further description of the antibodies or their properties is considered unnecessary.

[0073] In certain specific (but not 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 specific (but not limiting) embodiments, FITC antibodies are suitable for use herein, including FITC-mAB5405 available from Medix Biochemica (St. Louis, MO) and FITC-MAB130P available from BBI Solutions (Portland, ME). For example, Table 1 from Medix Biochemica demonstrates how commercially available anti-TSH antibodies 5401, 5404, 5405, 5407, 5408, and 5409 can be combined for use in sandwich immunoassays.

[0074] Table 1

[0075] In addition, non-limiting examples of antibodies that recognize TSH variants R55G / R75G include MAB130P from BBI Solutions (Portland, ME) (and particularly, but not limited to, FITC-MAB130P) and the antibody disclosed in U.S. Patent Application Publication No. 2025 / 0044304.

[0076] However, the various antibodies discussed above are not an exhaustive list of antibodies that can be utilized according to this disclosure; in fact, there are many other commercial sources of anti-TSH antibodies that can be utilized according to this disclosure. Therefore, those skilled in the art will be able to clearly and unambiguously identify and select the various anti-TSH antibodies that can be utilized according to this disclosure, and thus, further description of the anti-TSH antibodies or their characteristics is considered unnecessary.

[0077] In some non-limiting embodiments, suitable TSH biomarkers for use herein (and for which antibodies utilized according to this disclosure can specifically bind) include peptides comprising the following amino acid sequence: (SEQ ID NO:1), or a fragment or variant thereof, such as a variant having at least 95%, at least 97%, or at least 99% sequence identity with the biomarker sequence.

[0078] In some non-limiting embodiments, suitable biomarkers for use herein (and for which antibodies utilized according to this disclosure can specifically bind) include polypeptides comprising the following amino acid sequence: (SEQ ID NO:2), or a fragment or variant thereof, such as a variant having at least 95%, at least 97%, or at least 99% sequence identity with the biomarker. It should be noted that SEQ ID NO:2 is an R55G / R75G variant of TSH.

[0079] As used herein, the term "sequence identity" refers to the percentage of identical bases or amino acids determined by comparing a first polynucleotide or polypeptide with a second polynucleotide or polypeptide using algorithms with 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-ALIGN, etc.), which are available on websites 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 polynucleotide or polypeptide sequences is generally calculated using standard default parameters of various methods or computer programs.

[0080] Any antibody or antigen-binding fragment described herein or otherwise considered may be labeled or otherwise conjugated to various chemical or biomolecular parts for use in the diagnostic applications disclosed herein. Parts may be detectable labels, such as (but not limited to) chemiluminescent labels (e.g., acridinium esters and sulfonamides, luminol and isoluminol), phosphorescent labels, fluorescent labels (e.g., FITC), electrochemiluminescent labels (e.g., ruthenium(II) chelates), cloned enzyme donors, photosensitizer particles, or chemiluminescent particles for luminescent oxygen channel immunoassay (LOCI), lanthanide chelates for time-resolved fluorescence immunoassay (TR-FIA), radioactive... Labels, biotin, digoxigenin, enzymes, etc., such as radionuclides, including but not limited to tritium, carbon-14, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111, and fissile nuclides such as boron-10 or actinides, and any other detectable labeling / detection reagent disclosed herein or otherwise considered. In some non-limiting embodiments, the enzyme may be conjugated to the antibody for the purpose of detecting the bound antibody in a sample. Such enzyme conjugates include, but are not limited to, alkaline phosphatase (AP), horseradish peroxidase, β-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH). Those skilled in the art will understand that other enzymes used to determine antibody binding in solution-based immunoassays are also suitable as conjugates to the antibodies described herein. In addition, compounds such as acridinium esters can be conjugated to the provided antibodies to allow detection in immunoassays.

[0081] In certain specific (but not limiting) embodiments, the first label utilized in the first reagent of the kits, systems, and methods described herein or otherwise contemplated is a detectable label. For example (but not as a limitation), the first label may 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 are typically detectable only upon activation by a method, including but not limited to the addition of different chemicals, light stimulation, or exposure to a substrate or other compounds. When an acridinium ester compound is used, chemiluminescence is triggered by a peroxide and an acid / base, resulting in a flash that can be read by a suitable instrument. Additionally, an optional washing step may be used before initiating the detectability of the detectable label.

[0082] In one particular (but not limiting) embodiment, the first label is an acridine ester. In this case, the signal generation and detection steps of any method disclosed herein or otherwise considered may include the addition of at least one additional reagent that triggers chemiluminescence, which is quantified as relative light units (RLUs).

[0083] The second label utilized in the second reagent of the kits, systems, and methods described herein or otherwise considered can be any molecule, wherein an antibody or other specific binding partner against that molecule is available that can conjugate to a solid support, provided that the second label and antibody / binding partner do not otherwise interfere with the assay to be performed. Non-limiting examples of second label and antibody / specific binding partner combinations that may be used in kits, systems, and methods according to this disclosure include: fluorescein (e.g., fluorescein isothiocyanate (FITC)) for binding to an anti-fluorescein antibody or a fragment thereof; biotin for binding to an anti-biotin protein, streptoacidin, or an anti-biotin antibody; digoxigenin for binding to an anti-digoxigenin ligand antibody; other hapten and binding partner combinations; etc. These types of label and antibody / specific binding partner combinations are well known in the art and widely commercially available. Therefore, further description thereof is considered unnecessary.

[0084] Any solid support known in the art and capable of functioning according to the kits, systems, and methods described herein can be used in accordance with this disclosure. Non-limiting examples of usable solid supports include magnetic particles, such as (but not limited to) magnetic latex particles; paramagnetic particles (PMPs); cross-linked dextran available under the trade name SEPHADEX (Pharmacia FineChemicals, Piscataway, NJ); agarose; polystyrene beads; polyvinyl chloride; polystyrene; cross-linked polyacrylamide; nitrocellulose or nylon-based meshes such as sheets, strips, or paddles; or pores in tubes, plates, or microtiter plates (e.g., those prepared from polystyrene or polyvinyl chloride). When using paramagnetic particles, a magnetic field of some source can be used during an optional washing step to retain particles and molecules directly or indirectly bound to the particles. Molecules can be covalently bound by salt bridges, hydrogen bonds, or another type of bond.

[0085] The compositions / reagents of the kit or system may be provided in any form that allows them to function according to this disclosure. For example, but not as a limitation, each reagent may be provided in liquid form and disposed within the kit or system in bulk and / or as individual aliquots. Alternatively, in a particular (but not limiting) embodiment, one or more reagents may be disposed within the kit or system as individual aliquots of lyophilized reagents. The use of dried reagents in kits / microfluidic devices is described in detail in U.S. Patent No. 9,244,085.

[0086] Additionally, depending on the cross-reactivity and stability of the compositions / reagents, the compositions / reagents present in the kit or system may each be located in separate containers / compartments, or various compositions / reagents may be combined in one or more containers / compartments. Furthermore, the kit or system may include a device for disposing of one or more compositions / reagents (e.g., but not limited to, microfluidic devices or drug cartridges for loading onto clinical instruments).

[0087] In one particular (but not limiting) embodiment, a reagent comprising an anti-TSH variant antibody conjugated to a second label and a reagent comprising a solid support coated with an anti-second label antibody are combined in a single component / compartment within a kit.

[0088] The relative amounts of various compositions / reagents in a kit or system can be widely varied to provide composition / reagent concentrations that substantially optimize the reactions required to occur during the method, and further substantially optimize the sensitivity and selectivity of the assay. Where appropriate, one or more compositions / reagents in the kit or system may be provided as dry powders, such as lyophilized powders, and the kit or system may further include excipients for dissolving the dried reagents; in this way, a reagent solution of appropriate concentration can be obtained from these compositions for performing the method or assay according to this disclosure. Quality control and / or calibration reagents may also be included in the kit or system. Additionally, the kit or system may further include a set of written instructions (or electronic instruction access) explaining how to use the kit or system. Kits or systems of this nature can be used for any method described herein or otherwise considered.

[0089] Under the European Union REACH regulations, Triton X-100 nonionic surfactant is classified as a "Substance of Very High Concern (SVHC)," and therefore its use and sale within the EU are prohibited without specific authorization. Consequently, in some non-limiting embodiments, all reagents utilized according to this disclosure do not contain Triton X-100, but instead contain another nonionic surfactant not subject to REACH regulations, such as (but not limited to) TERGITOL™ 15-S-9 (Dow Chemical Company).

[0090] In some non-limiting embodiments, this disclosure facilitates the immediate detection or remote diagnosis of thyroid disease or pituitary disorders and assists healthcare providers in monitoring the status or progression of thyroid disease or pituitary disorders (and / or their treatment) at two or more time points. Importantly, this disclosure provides healthcare decision-makers with an alternative to potentially inaccurate measurements.

[0091] In some non-limiting embodiments, this disclosure employs a computer-implementable algorithmic approach that utilizes one or more TSH-related marker values. The predictive value of this disclosure has been validated in clinical studies to monitor the status or progression of thyroid disease or pituitary disorders.

[0092] In some non-limiting embodiments, analytical methods are applied to information derived from TSH measurements according to this 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, this disclosure provides a data structure stored in a computer-readable medium, which can be read by a microprocessor and includes at least one code that uniquely identifies a linear or nonlinear function algorithm derived in the manner described herein.

[0094] In another non-limiting embodiment, this disclosure provides a diagnostic kit comprising: (a) a data structure stored in a computer-readable medium, readable by a microprocessor and including at least one code that uniquely identifies a linear or nonlinear function algorithm derived in the manner described herein; and (b) one or more immunoassays that detect and determine serum marker values ​​of a patient.

[0095] The methods and kits provided herein can detect TSH biomarkers in samples with sufficient sensitivity and specificity, making the test results reliable enough for medical action. The methods and kits described herein for detecting and / or diagnosing thyroid disease in subjects detect TSH or its variants with a sensitivity greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100%. In some non-limiting embodiments, the methods and kits provided herein can detect TSH or its variants with a sensitivity of about 70%-100%, about 80%-100%, or about 90%-100%. In some non-limiting embodiments, the methods and kits provided herein can detect TSH or its variants with a sensitivity and specificity of about 50%-100%, about 60%-100%, about 70%-100%, about 80%-100%, or about 90%-100%.

[0096] Any method described herein or otherwise considered can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some non-limiting embodiments, the computer system may be configured to output a TSH biomarker score based on the levels of two or more of the received TSH biomarker profile and / or any TSH biomarkers disclosed herein or otherwise considered. In particular, the computer program may include instructions for the system to select appropriate subsequent steps, including additional medication, treatment, and / or additional testing for the subject.

[0097] In some non-limiting embodiments, the computer program may be configured to enable the computer system to identify subjects for further testing (e.g., thyroid disease testing) based on received data (e.g., TSH biomarker profile), identify subjects as being at risk of or having thyroid disease or pituitary disease, and / or identify subjects to receive medication, and use the data to calculate a TSH biomarker score. The system may be able to rank subsequent steps of identification based on the TSH biomarker profile, accompanied by demographic factors and / or image-based biomarkers. The system may be able to adjust the ranking based on, for example, the clinical response of the subject or a family member of the subject (who has or is suspected of having thyroid disease).

[0098] Figure 2 This is a block diagram of a non-limiting embodiment of a computer system 1100 that can be used in the operations described above. System 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Components 1110, 1120, 1130, and 1140 are interconnected via a system bus 1150. The system may include an analytical device 1160 for determining the levels of one or more biomarkers of this disclosure in a sample.

[0099] In some non-limiting embodiments, processor 1110 is capable of processing instructions for execution within system 1100. In one non-limiting embodiment, processor 1110 is a single-threaded processor. In another non-limiting embodiment, processor 1110 is a multi-threaded processor. Processor 1110 is capable of processing instructions stored in memory 1120 or on storage device 1130, including receiving or transmitting information via input / output device 1140.

[0100] In some non-limiting embodiments, memory 1120 stores information within system 1100. In one non-limiting embodiment, memory 1120 is a computer-readable medium. In one non-limiting embodiment, memory 1120 is a volatile memory cell. In another non-limiting embodiment, memory 1120 is a non-volatile memory cell.

[0101] Storage device 1130 is capable of providing massive storage for system 1100. In one non-limiting embodiment, storage device 1130 is a computer-readable medium.

[0102] Input / output device 1140 provides input / output operations for system 1100. In one non-limiting embodiment, input / output device 1140 includes a keyboard and / or pointing device. In one non-limiting embodiment, input / output device 1140 includes a display unit for displaying a graphical user interface.

[0103] Furthermore, certain non-limiting embodiments of this disclosure relate to a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the methods provided herein. For example, Figure 3 and Figure 4 The document illustrates a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including those described herein. In some non-limiting embodiments, the non-transitory computer-readable medium includes hard disks, external hard disks, optical disks, CDs, DVDs, etc., for storing data. In some non-limiting embodiments, software disposed within a physical medium is also suitable for use herein.

[0104] In some non-limiting embodiments, a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including methods for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary syndrome in an individual. The method includes the steps of: (a) incubating a biofluid sample with two or more antibodies (at least one binding to wild-type TSH and at least one binding to a TSH variant) that specifically bind to TSH biomarkers, under conditions allowing the formation of at least two antibody-TSH immune complexes; (b) measuring the amount of the formed antibody-TSH immune complexes to obtain a measurement of TSH in the sample; and (c) using a mathematical algorithm to obtain a TSH score based on the measurement of TSH in the sample. In some non-limiting embodiments, incubating the biofluid sample includes incubating with two or more antibodies that specifically bind to TSH and highly correlated TSH variants under conditions allowing the formation of antibody-TSH immune complexes.

[0105] Now for reference Figure 3 The figure depicts a method 1200 for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorders in an individual in accordance with this disclosure.

[0106] Now for reference Figure 4 The figure depicts a method 1300 for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorders in an individual in accordance with this disclosure. Example

[0107] Examples are provided below. However, this disclosure should be understood as not being limited in its application to the specific experiments, results, and laboratory procedures disclosed herein. Rather, the examples are provided only as one of various implementations and are intended to be exemplary rather than exhaustive.

[0108] Example 1 Figure 1 A non-limiting embodiment of a total TSH assay constructed according to this disclosure is depicted graphically, wherein the total TSH assay detects both wild-type TSH (WT TSH) and TSH variant R75G in a biological sample. The assay utilizes the following components: (i) Auxiliary well reagent containing a FITC-labeled mouse monoclonal antibody (FITC-mAB5405) that specifically binds to WT TSH; (ii) A solid-phase reagent comprising paramagnetic particles coated with anti-FITC antibody (PMP::anti-FITC) and to which a FITC-labeled antibody (FITC-mAB130P) for detecting TSH R75G has been added; and (iii) Lite reagent, which contains a Fab fragment of another anti-TSH antibody that binds to both WT TSH and TSH R75G and is labeled with acridinium ester (Fab5409::BSA::HEGAE).

[0109] The assay was also performed in the presence of 3 g / L TERGITOL™ 15-S-9 (Dow Chemical Company).

[0110] In one non-limiting embodiment of the assay method, 100 µL of biological sample is mixed with 50 µL of auxiliary well reagent and 50 µL of Lite reagent, and incubated for approximately 2.75 minutes. Then, 200 µL of solid-phase reagent is added, and the mixture is incubated for 5.5 minutes. A washing step is then performed, and acid and base reagents are added to initiate the chemiluminescent reaction; the amount of relative optical units (RLU) generated is subsequently detected by the system.

[0111] Example 2 New and improved TSH assays and kits are available for use with clinical analyzers such as (but not limited to) ATELLICA. ® The IM Analyzer (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) is used for the in vitro diagnostic determination of thyroid-stimulating hormone (TSH) in biological samples such as (but not limited to) human serum and plasma (EDTA and lithium heparin). Measurement of TSH produced by the anterior pituitary gland is used for the diagnosis of thyroid or pituitary disorders.

[0112] Thyroid-stimulating hormone (TSH) is a glycoprotein with two non-covalently bound subunits. The α subunit is similar to those of follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and luteinizing hormone (LH). The β subunit of TSH is unique, contributing to the hormone's specific biochemical and immunological properties. The TSH response involves a negative feedback mechanism of FT3 (free T3) and FT4 (free T4) concentrations, synthesized and secreted by the anterior pituitary gland. Additionally, the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), directly stimulates TSH production. TSH interacts with specific cellular receptors on the surface of thyroid cells and plays two main roles: first, stimulating cell proliferation and hypertrophy; and second, stimulating the thyroid gland to synthesize and secrete T3 and T4.

[0113] The ability to quantify circulating TSH levels is important in assessing thyroid function. It is particularly useful for differentiating primary (thyroid) hypothyroidism from secondary (pituitary) and triple (hypothalamic) hypothyroidism. In primary hypothyroidism, TSH levels are significantly elevated, while in secondary and triple hypothyroidism, TSH levels are lower. TRH stimulation differentiates secondary and triple hypothyroidism by observing changes in TSH levels in patients. Typically, the TSH response to TRH stimulation is absent in secondary hypothyroidism and normal to excessive in triple hypothyroidism. Historically, TRH stimulation has been used to confirm primary hyperthyroidism, indicated by elevated T3 and T4 levels and low or undetectable TSH levels. TSH measurement with increased sensitivity and specificity provides a primary diagnostic tool for differentiating patients with hyperthyroidism from those with euthyroidism.

[0114] In one non-limiting embodiment, the assay employs a FITC-labeled mouse monoclonal anti-wild-type TSH antibody, a FITC-labeled mouse monoclonal anti-TSH variant antibody, a mouse monoclonal anti-fluorescein antibody linked to paramagnetic particles, and a tracer for chemiluminescent detection comprising an acridinium ester (AE)-labeled anti-TSH mouse monoclonal antibody conjugated to bovine serum albumin (BSA), wherein the AE-labeled antibody can bind to both TSH and the TSH variant, thereby the binding of either the FITC-labeled antibody and the AE-labeled antibody results in the formation of a sandwich immune complex (one sandwich immune complex containing TSH and another sandwich immune complex containing the TSH variant). There is a direct relationship between the amount of TSH present in the patient sample and the amount of relative optical units (RLU) detected by the system.

[0115] This assay can be used with any biological sample, such as (but not limited to) serum and plasma (EDTA and lithium heparin). In one specific (but not limiting) embodiment, the assay requires at least 75 µL of sample for a single assay; this volume does not include unavailable volume in the sample container or additional volume required to perform repeat assays or other tests on the same sample. Do not use samples with significant contamination. Ensure the sample is free of air bubbles, foam, fibrin, or other particulate matter before placing it on the system. Remove particulates by centrifugation, following CLSI guidelines and the recommendations of the collection device manufacturer.

[0116] In one non-limiting embodiment, the materials present in the main reagent kit include: • Lite Reagent (6.00 mL / pack) - BSA conjugated with mouse monoclonal anti-TSH antibody (~0.3 µg / mL) in a buffer containing mouse IgG labeled with acridinium ester; BSA; bovine gamma globulin (BGG); goat serum; sodium azide (<0.1%); surfactant; and preservative. • Solid phase (21.0 mL / packet) – FITC-labeled mouse monoclonal anti-TSH variant antibody and mouse monoclonal anti-fluorescein antibody (85 µg / mL) linked to paramagnetic particles in buffer; BSA; BGG; goat serum; sodium azide (<0.1%); surfactant; preservative. The anti-TSH variant antibody binds to at least one variant, such as (but not limited to) the R55G / R75G TSHB variant. • Auxiliary well reagents (6.0 mL / packet) - FITC-labeled mouse monoclonal anti-wild-type TSH antibody (~3 µg / mL); BSA; BGG; goat serum; sodium azide (<0.1%); surfactant; preservative.

[0117] In this way, the assays and kits disclosed herein are able to accurately measure TSH levels in populations of rare genetic variants.

[0118] The kit disclosed herein may further include at least one calibrator containing a known concentration of TSH. In certain specific (but not limiting) embodiments, the kit may include a "low" calibrator and a "high" calibrator, wherein the low calibrator has a lower TSH concentration than the high calibrator. The calibrator may contain wild-type TSH and / or TSH variants.

[0119] In one non-limiting embodiment, the assays and kits disclosed herein are used on a clinical analyzer system that performs the following steps (automatically or manually): 1. Dispense 75 µL of sample into a cuvette. 2. Dispense 38 µL of auxiliary well reagent and 38 µL of Lite reagent, and then incubate at 37°C for 5 minutes. 3. Distribute 150 µL of solid phase and incubate at 37°C for 7 minutes. 4. Use a washing agent (e.g., but not limited to, ATELLICA) ® IM Wash (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) performs the washing sequence. 5. Dispense 300 µL each of the acid and base reagents (e.g., but not limited to, ATELLICA). ® IM Acid Reagent and ATELLICA ® IM Base Reagent is used to initiate a chemiluminescent reaction. 6. Report the results.

[0120] The system uses the calculation procedure described in the system's online help to determine the results. Depending on the units defined when setting up the assay, the system reports results in pIU / mL (common units) or mIU / L (SI units). The conversion formula is 1 pIU / mL = 1 mIU / L. The results of this assay should always be interpreted in conjunction with the patient's medical history, clinical presentation, and other findings (if any).

[0121] The predicted value is used in ADVIA CENTAUR ® ADVIA CENTAUR on the system (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) ® The existing technology TSH3-UL measurement was used to determine this, and the results were confirmed through comparative measurements. ATELLICA® The reagent formulations used on the IM Analyzer (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) are similar to ADVIA CENTAUR. ® The same ones used on the system.

[0122] According to CLSI document EP28-A3c, the reference range for healthy (normal thyroid function) adults uses ADVIACENTAUR. ® ADVIA CENTAUR on the system ® The existing technology TSH3-UL determination was used, and ATELLICA was used. ® ATELLICA on IMAnalyzer ® The new total TSH measurement of IM was validated.

[0123] Samples were measured for TSH, FT3, and FT4, and were considered normal if their values ​​were within acceptable ranges. Samples were also screened for the presence of thyroid autoantibodies (Kratsch et al. (2008) Clin Biochem, 41(13): 1091-1098). Reference intervals are shown in Table 2 and were determined by calculating the 2.5th and 97.5th percentiles of the value distribution.

[0124] Table 2 a Number of test samples.

[0125] According to CLSI document EP28-A3c, the reference range for healthy pediatric populations (infants, children, and adolescents) uses ADVIA CENTAUR. ® ADVIA CENTAUR on the system ® The existing technology TSH3-UL measurement was used to determine this.

[0126] Using predetermined inclusion criteria, samples were prospectively collected from pediatric subjects who were presently healthy (with normal thyroid function). Reference intervals were established for children and adolescents using nonparametric methods, where the 2.5th and 97.5th percentiles of the value distribution were calculated. For the infant population, methods that accommodated smaller sample sizes were used to calculate reference intervals.

[0127] Performance characteristics: In ATELLICA ® The reagents used in IM Analyzer are compatible with ADVIA CENTAUR. ®The same applies to those used on the system. Regarding ATELLICA. ® Some performance characteristics measured by IM were obtained using ADVIA CENTAUR ® The system will determine this.

[0128] The measurement range is 0.010–150.000 pIU / mL (mIU / L). The lower limit of the measurement range is defined by the limit of quantitation (LoQ). Results below the measurement range are reported as <0.010 pIU / mL (mIU / L).

[0129] Detection capabilities are defined as follows: Limit of Blank (LoB) - 0.005 pIU / mL (mIU / L); Limit of Detection (LoD) - 0.008 pIU / mL (mIU / L); and Limit of Quantification (LoQ) - 0.010 pIU / mL (mIU / L). Detection capabilities are determined according to CLSI document EP17-A2. LoB corresponds to the highest measurement result that is most likely to be observed with a 95% probability for a blank sample. LoD corresponds to the lowest analyte concentration that can be detected with a 95% probability. LoQ corresponds to the lowest analyte concentration with a laboratory CV ≤20%.

[0130] Accuracy was determined according to CLSI document EP05-A3. A 20-day protocol was used, with samples measured in two replicates, and two runs per day.

[0131] The results shown in Table 3 are representative of the measured performance. The measurement design was carried out according to CLSI document EP05-A3, using a 20-day protocol, and achieved the accuracy shown in Table 4.

[0132] Table 3 a Number of measurements. b Standard deviation. c Coefficient of variation.

[0133] Table 4

[0134] Reproducibility was determined according to CLSI document EP05-A3. Tests were performed using three instruments and three reagent batches. A 5-day protocol was used, with samples measured in five replicates, one run per day (225 measurements per sample).

[0135] The results shown in Table 5 are representative of the measured performance. The measurement design was carried out according to CLSI document EP05-A3, and when using a 5-day protocol, it exhibits the reproducibility shown in Table 6.

[0136] Table 5 a Standard deviation. b Coefficient of variation.

[0137] Table 6

[0138] According to CLSI document EP09c-ed3, the measurement comparisons were determined using a Passing-Bablok regression model. The results are shown in Table 7. The consistency of measurements can vary depending on the study design, the comparative measurements, and the test population. The measurement design was configured with a correlation coefficient ≥0.97 and a slope of 1.0 ± 0.075.

[0139] Table 7 a Number of test samples. b Coefficient of variation.

[0140] According to CLSI document EP09c-ed3, use ADVIA CENTAUR ® The XP system was used to determine sample equivalence using a Passing-Bablok regression model. The results are shown in Table 8. The consistency of sample types can vary depending on the study design and the test population. The assay design was designed with a correlation coefficient ≥ 0.95 and a slope of 0.95 ± 1.05.

[0141] Table 8 a Number of test samples. b Coefficient of variation.

[0142] Linearity testing was performed according to CLSI document EP06-ed2. The determination was linear over the measurement range of 0.010–150.000 µIU / mL (mIU / L).

[0143] High TSH concentrations can cause an abnormal decrease in RLU (high-dose hook effect). In this assay, patient samples with TSH concentrations above the measurement range and up to 3000 µIU / mL will be reported as >150 µIU / mL (mIU / L).

[0144] The standardization of the assay can be traced back to the World Health Organization (WHO) third-generation human TSH international reference formulation (3). rd International Reference Preparation for human TSH) (IRP81 / 565).

[0145] Example 3 Background Overview: Inconsistent results have been reported for thyroid-stimulating hormone (TSH) assays from various manufacturers in patients with the R75G mutation in the TSH β chain. Although functional, TSH with this mutation (TSH R75G) exhibits altered immunoreactivity, rendering it undetectable by some assays, such as (but not limited to) Siemens Healthineers' TSH3-Ultra (TSH3-UL) assay. A novel total TSH assay of this embodiment was developed to address this issue. A methodological comparison study was conducted using the developed assay and a reference assay (i.e., Siemens Healthineers' TSH assay, which accurately measures TSH R75G).

[0146] Methods Overview: Fifty-nine natural human serum samples containing the TSH R75G mutation from two laboratories in India and the United States were analyzed. PCR and DNA sequencing confirmed the R75G mutation in all samples. To measure TSH R75G, two replicates of each sample were analyzed using existing TSH assays and a novel total TSH assay.

[0147] Results Summary: The mean TSH concentration measured by the new total TSH assay ranged from 0.874 to 23.209 μIU / mL. This range is within the measured range (0.010–150.000 µIU / mL) and is comparable to the mean concentration measured by the prior art TSH assay (range, 0.850–22.536 μIU / mL). The TSH R75G concentrations in individual samples measured by the new total TSH assay were similar to those measured by the prior art TSH assay. 53 samples had TSH concentrations ≤10 μIU / mL; 6 samples had TSH concentrations >10 μIU / mL. Passing-Bablok regression analysis indicated that the new total TSH assay was performed comparablely to the prior art TSH assay. Bland-Altman plots provided confirmation of the results.

[0148] Conclusion Summary: The assay disclosed herein can accurately and precisely measure TSH R75G in natural human serum.

[0149] introduction Measurement of circulating thyroid-stimulating hormone (TSH) concentrations is crucial for investigating thyroid dysfunction, monitoring levothyroxine therapy, and monitoring therapy for hyperthyroidism (Van Uytfanghe et al. (2023)). Thyroid ® . 33(9):1013-1028). Inaccurate readings by TSH measurement can lead to inaccurate diagnosis and / or inappropriate treatment.

[0150] Previous reports have described inconsistent TSH assay results in patients with the R75G mutation in the β chain of TSH (TSH R75G), also known as the R55G mutation (R55G nomenclature based on the human genome assembly GRCh37.p13:chr1:115,576,654 A>G (2013), and R75G nomenclature based on the more recent human genome assembly GRCh38.p14:chr1:115,034,033 A>G (2022). See Genome Reference Consortium. Human Genome Overview - GRCh37.p13, 2013; and Genome Reference Consortium. Human Genome Overview - GRCh38.p14, 2022. Some manufacturers’ TSH assays are able to accurately measure the concentration of TSH R75G, while certain Siemens Healthineers TSH assays on multi-instrument platforms cannot detect and measure TSH with this mutation (Drees et al., J ClinEndocrinolMetab. 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; 11(1)). Further investigation revealed that TSH R75G is functional, but its immunoreactivity is altered, making it undetectable by solid-phase monoclonal antibody (mAb) in Siemens Healthineers’ TSH3-Ultra (TSH3-UL) assay (Drees et al., introduced above).

[0151] To address the inability of the TSH3-UL assay to detect TSH R75G, the assay disclosed herein was developed. Unlike its predecessors, the assay disclosed herein contains an additional mAb for the detection of TSH R75G. This embodiment reports the results of a comparative study of TSH R75G measurement using the new assay and a reference method (i.e., the prior art TSH assay of Siemens Healthineers, which utilizes a polyclonal capture antibody) previously shown to accurately measure TSH R75G (Drees et al., cited above).

[0152] Materials and Methods This comparative study was conducted according to CLSI EP09c-ED3 (CLSI. Measurement Procedure Comparison and Bias Estimation Using Patient Samples. 3rd Edition. CLSI guideline EP09c. Wayne, PA: Clinical and Laboratory Standards Institute; 2018). The difference between the two TSH assays is that the existing TSH assay (Siemens Healthineers, Inc.) has a solid-phase sheep polyclonal antibody against TSH and a lite-reagent mouse mAb against TSH, while the current assay uses a fluorescein isothiocyanate (FITC)-labeled mouse mAb against a TSH variant, a FITC-labeled anti-wild-type TSH-captured mouse mAb, a mouse anti-fluorescein antibody linked to paramagnetic particles, and a tracer for chemiluminescent detection, which consists of a proprietary acridinium ester and an anti-TSH mouse mAb conjugated to bovine serum albumin.

[0153] Seventy-four natural human serum samples containing TSH R75G were generously provided by Dr. Julia Drees (Kaiser Permanente Northern California Regional Laboratory, Richmond, CA) and Dr. Nimmi Kansal (Dr. Lal PathLabs, Delhi, India). These samples were selected based on previous analyses that showed TSH results were lower than those obtained with ADVIA CENTAUR. ® XP or ATELLICA ®The IM assay has the limit of quantitation (LoQ) of the existing TSH3-UL assay, but is higher than the LoQ of other existing TSH assays, including those using polyclonal antibodies. All samples were received in accordance with the Material Transfer Agreement (MTA) between Siemens Healthineers and the respective laboratories.

[0154] The presence of the R75G mutation was confirmed by PCR and DNA sequencing (Antibody Systems, Inc., Hurst, TX USA). DNA was extracted from blood clots from Dr. Lal PathLabs using the QIAamp DNA Blood Micro-Test Kit (QIAGEN, Germantown, MD), following the manufacturer's protocol. The DNA was then used with the Phusion High Fidelity PCR Kit (Thermo Fisher Scientific Inc., Waltham, MA) and... TSHB PCR amplification was performed using specific forward (5'-TGTTTCCTAAAGTCCTGTCAC-3'; SEQ ID NO:3) and reverse (5'-GCTTTATTTCAGGCAAGCAC-3'; SEQ ID NO:4) primers (IDT Technologies, Coralville, IA). Products underwent Sanger sequencing (GENEWIZ, South Plainfield, NJ) using the same primers used for amplification. The human genome assembly GRCh38.p14 was used as a reference genome, and position chr1:115,034,033 served as a reference point for mutations. Sequences were analyzed using SnapGene version 6.2.1 (GENEWIZ). For all samples from Dreees's laboratory, the R75G mutation had previously been confirmed by PCR and DNA sequencing (Drees et al., introduced above).

[0155] via ADVIA CENTAUR ® XP TSH assay and the new total TSH assay format were used to test and measure the TSH content of the sample using the ADVIA CENTAUR method. Following the manufacturer's instructions, the test was performed at ADVIA CENTAUR. ®On the XP system, two replicates were performed for each TSH assay, kit-specific calibrator, standard, and commercial control (Bio-Rad Lyphochek Immunoassay PlusControl, Hercules, CA). Data reduction and analysis were performed using Raven Calculation software version 4 (Siemens Healthineers Diagnostics, Tarrytown, New York), with one-sided 95% confidence intervals using Passing-Bablok linear regression.

[0156] Results and Discussion Of the 74 natural serum samples with confirmed TSH R75G, 15 were excluded from further analysis due to insufficient volume for two replicates per assay. The mean TSH concentration in the remaining 59 samples, as measured by the new total TSH assay, ranged from 0.874 to 23.209 μIU / mL (mIU / L) (Table 9). This range is within the measurement interval for this assay (0.010–150.000 µIU / mL [mIU / L]) and is comparable to that measured by existing technology TSH assays using polyclonal antibodies (mean, 0.850–22.536 μIU / mL [mIU / L]).

[0157] Individual sample comparisons showed that the TSH R75G concentrations measured by the new total TSH assay were similar to those measured by the reference TSH assay using polyclonal antibodies (Table 9). Most samples had concentrations ≤10 μIU / mL (TSH concentrations <2 μIU / mL for 21 samples; 2–5 μIU / mL for 24 samples; and 5–10.0 μIU / mL for 8 samples), and 6 samples had TSH concentrations >10 μIU / mL.

[0158] Table 9 a All samples except LSam8 were homozygous for the R55G mutation in the TSH β chain, and the TSH concentration of all samples except LSam8 was <0.01 µIU / mL, as measured by TSH3UL assay. b Sample LSam8 is heterozygous for the R55G mutation in the TSH β chain.

[0159] The only sample heterozygous for TSH R75G (sample LSam8) had a TSH concentration of 3.62 µIU / mL as measured by TSH3-UL assay (measurement range, 0.008–150.0 µIU / mL); however, the TSH concentrations detected by both assays were approximately twice that determined by TSH3-UL assay (7.741 and 7.904 µIU / mL, respectively). A possible explanation for this result is equivalent expression of two TSH alleles (including one allele related to TSH R75G) and the detection of only TSH R75G (or approximately 50% of total TSH) by TSH3UL.

[0160] Passing-Bablok regression analysis using serum samples with TSH R75G indicated that the new total TSH assay was comparable to the old TSH assay performed with polyclonal antibodies. Figure 5 (See small figure A). The Bland-Altman plot provides the validation results. Figure 5 (See Figure B), but the two outlier samples exhibited a bias between -40% and -60%. The absolute difference observed between the two replicates of each outlier TSH R75G sample ranged from 0.123 to 0.253 µIU / mL for existing TSH assays (using polyclonal antibodies) and from 0.001 to 0.008 µIU / mL for the new total TSH assay. Therefore, individual replicate outliers are not the cause of the bias. Furthermore, interfering substances (e.g., hemoglobin, triglycerides, and cholesterol) were not identified as a cause. The observed bias is most likely due to sample specificity issues.

[0161] Outliers were observed in wild-type (n=2) and R75G (n=2) TSH serum samples, with the magnitude of the deviation being similar between wild-type and mutant samples.

[0162] in conclusion Our results demonstrate that the novel total TSH assay described herein can accurately and precisely measure TSH R75G in natural human serum. Therefore, this assay can be effectively used to evaluate TSH in patients who may be at risk of having an R75G mutation in the TSH β chain.

[0163] Non-restrictive illustrative implementation scheme Illustrative Embodiment 1. A kit for performing an immunoassay for thyroid-stimulating hormone (TSH), the kit comprising: a first reagent containing a first label conjugated to a first anti-TSH monoclonal antibody or an antigen-binding fragment thereof; a second reagent containing a second label conjugated to a second anti-TSH monoclonal antibody or an antigen-binding fragment thereof; a third reagent containing a third anti-TSH monoclonal antibody or an antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof is conjugated to the second label; a fourth reagent containing a label specifically binding to the first anti-TSH monoclonal antibody or an antigen-binding fragment thereof. A solid support coated with a second labeled antibody or its antigen-binding fragment; wherein the first and second anti-TSH monoclonal antibodies or their antigen-binding fragments bind substantially non-overlapping epitopes of TSH to form a sandwich complex, and the antibody of the fourth reagent or its antigen-binding fragment specifically binds 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 their antigen-binding fragments bind substantially non-overlapping epitopes of TSH variants to form a sandwich complex, and the antibody of the fourth reagent or its antigen-binding fragment binds the second label of the third reagent to attach the sandwich complex to the solid support.

[0164] Illustrative Embodiment 2. The kit of Illustrative Embodiment 1, wherein the first label is selected from chemiluminescent compounds, phosphorescent compounds, fluorescent compounds, radioactive labels, biotin, and enzymes.

[0165] Illustrative Implementation Scheme 3. The kit of Illustrative Implementation Scheme 2, wherein the first label is acridine ester.

[0166] Illustrative Embodiment 4. A kit of any one of Illustrative Embodiments 1-3, wherein the second label is selected from fluorescein, streptoacidin, anti-biotin antibody or its antigen-binding fragment, and digoxigenin.

[0167] Illustrative Embodiment 5. The kit of Illustrative Embodiment 4, wherein the second label is fluorescein, and wherein the third antibody or its antigen-binding fragment is an anti-fluorescein monoclonal antibody or its antigen-binding fragment.

[0168] Illustrative Embodiment 6. The kit of Illustrative Embodiment 5, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0169] Illustrative Embodiment 7. A kit of any one of Illustrative Embodiments 1-6, wherein the solid support comprises paramagnetic particles.

[0170] Illustrative Embodiment 8. A kit of any one of Illustrative Embodiments 1-7, wherein the TSH variant has at least 99% sequence identity with natural TSH.

[0171] Illustrative Embodiment 9. A kit of any one of Illustrative Embodiments 1-8, wherein the sequence of the TSH variant differs from the sequence of the native TSH by one amino acid.

[0172] Illustrative Embodiment 10. A kit for any one of Illustrative Embodiments 1-9, wherein the TSH variant specifically bound to the third anti-TSH monoclonal antibody or its antigen-binding fragment is R75G.

[0173] Illustrative Embodiment 11. A kit of any one of Illustrative Embodiments 1-10, wherein none of the first, second, third and fourth reagents contains octylphenol polyoxyethylene ether.

[0174] Illustrative Embodiment 12. A kit of any one of Illustrative Embodiments 1-11, wherein at least one of the first reagent, the second reagent, the third reagent, and the fourth reagent comprises nonylphenol polyoxyethylene ether.

[0175] Illustrative Implementation Scheme 13. A kit of any one of Illustrative Implementation Schemes 1-12, wherein the third reagent and the fourth reagent are combined as a single component within the kit.

[0176] Illustrative Embodiment 14. A system for performing an immunoassay for TSH, the system comprising: a kit of any one of Illustrative Embodiments 1-13; and a sample collection tube.

[0177] Illustrative Embodiment 15. The system of Illustrative Embodiment 14, wherein the sample collection tube is a serum separation tube.

[0178] Illustrative Embodiment 16. The system of Illustrative Embodiment 14 or 15, further comprising at least one additional reagent for initiating the first label detection.

[0179] Illustrative Embodiment 17. A system of any one of Illustrative Embodiments 14-16, wherein the first label comprises an acridinium ester, and wherein the at least one additional reagent initiates chemiluminescent detection triggered by the acridinium ester.

[0180] Illustrative embodiment 18. The system of illustrative embodiment 16 or 17, wherein the at least one additional reagent comprises an acid and a base.

[0181] Illustrative Embodiment 19. A method for determining the total TSH concentration in a biological sample, the method comprising the steps of: (1) simultaneously or wholly or partially sequentially combining (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 an antigen-binding fragment thereof; (c) a second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or an antigen-binding fragment thereof; (d) a third reagent comprising a third anti-TSH monoclonal antibody or an antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof is conjugated to the second label; (e) a fourth reagent comprising a solid support coated with an antibody or an antigen-binding fragment thereof that specifically binds to a second label; (2) incubating the mixture under conditions in which (b) and (c) bind to TSH present in the biological sample to form a first sandwich complex, (b) and (d) bind to a 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 sandwich complex and the second sandwich complex to the solid support of (e); (3) detecting the signal generated by the first label indirectly bound to the solid support of (e); and (4) determining the 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.

[0182] Illustrative Embodiment 20. The method of Illustrative Embodiment 19, wherein the biological sample is selected from blood, serum, plasma, saliva, sputum, mucus, nose, nasopharynx, anterior nose, oropharynx, trachea, bronchoalveolar and combinations thereof.

[0183] Illustrative Embodiment 21. The method of Illustrative Embodiment 19 or 20 further includes, prior to step (1), performing a step of separating a biological sample using at least one sample collection tube.

[0184] Illustrative Embodiment 22. The method of Illustrative Embodiment 21, wherein the sample collection tube is a serum separation tube.

[0185] Illustrative Implementation Scheme 23. The method of any one of Illustrative Implementation Schemes 19-22, further comprising performing a washing step prior to step (3).

[0186] Illustrative Embodiment 24. A method of any one 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, the chemiluminescence being quantified as relative light units (RLU).

[0187] Illustrative Embodiment 25. The method of any one 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 an antifluorescein monoclonal antibody or antigen-binding fragment thereof.

[0188] Illustrative Implementation Scheme 26. The method of any one of Illustrative Implementation Schemes 19-25, wherein the solid support of (c) comprises paramagnetic particles.

[0189] Illustrative Embodiment 27. The method of any one of Illustrative Embodiments 19-26, wherein the third anti-TSH monoclonal antibody or its antigen-binding fragment specifically binds to a TSH variant that has at least 99% sequence identity with native TSH.

[0190] Illustrative Embodiment 28. The method of any one of Illustrative Embodiments 19-27, wherein the sequence of the third anti-TSH monoclonal antibody or the TSH variant to which its antigen-binding fragment specifically binds differs from the sequence of native TSH by one amino acid.

[0191] Illustrative Embodiment 29. The method of any one of Illustrative Embodiments 19-28, wherein the TSH variant specifically bound to the third anti-TSH monoclonal antibody or its antigen-binding fragment in (d) is R75G.

[0192] Illustrative Embodiment 30. The method of any one of Illustrative Embodiments 19-29, wherein the method is performed in the absence of octylphenol polyoxyethylene ether.

[0193] Illustrative Embodiment 31. The method of any one of Illustrative Embodiments 19-30, wherein at least one of (b)-(e) comprises nonylphenol polyoxyethylene ether.

[0194] Illustrative Implementation Scheme 32. The method of any one of the illustrative implementation schemes 19-31, wherein (b)-(e) each comprises nonylphenol polyoxyethylene ether.

[0195] Illustrative Implementation Scheme 33. The method of any one of the illustrative implementation schemes 19-32, further defined as a method for determining the presence, severity and / or susceptibility to thyroid disease or pituitary disorder in an individual.

[0196] Illustrative Implementation 34. A method of any one of Illustrative Implementations 19-33, wherein step (4) is further defined as comprising the following steps: (i) measuring the amount of the first sandwich complex and the second sandwich complex formed to obtain a measurement of total TSH (total TSH + TSH variant) in the biological sample; and (ii) using a mathematical algorithm to obtain a TSH score based on the measurement of total TSH in the sample.

[0197] Illustrative Implementation Scheme 35. The method of Illustrative Implementation Scheme 34, wherein the TSH score is used to support, predict, or replace thyroid testing.

[0198] Illustrative Implementation Scheme 36. The method of Illustrative Implementation Scheme 34 or 35, wherein the mathematical algorithm is a discriminant function algorithm.

[0199] Illustrative Implementation Scheme 37. The method of Illustrative Implementation Scheme 36, wherein the discriminant function algorithm is a linear discriminant function algorithm.

[0200] Illustrative Embodiment 38. A method for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorder in an individual, the method comprising the steps of: (a) obtaining a biological fluid sample from the individual; (b) incubating the biological fluid sample with at least one antibody that specifically binds to wild-type TSH and at least one antibody that specifically binds to at least one TSH variant (e.g., but not limited to TSH R75G) under conditions that allow the formation of one or more antibody-TSH immune complexes; (c) measuring the amount of antibody-TSH immune complexes formed to obtain a measurement of total TSH (TSH + TSH variant) in the sample; and (d) using a mathematical algorithm to obtain a TSH score based on the measurement of total TSH biomarkers in the sample.

[0201] Illustrative Implementation 39. A method for determining the presence, severity, and / or susceptibility to a disease in an individual, the method comprising the steps of: (a) obtaining a biological fluid sample from the individual; (b) selecting at least two diagnostic markers for the disease from the sample, wherein the at least two diagnostic markers are selected from TSH, TSH variants, or peptides having 99% sequence identity with TSH; (c) measuring the amount of each of the at least two diagnostic markers in the sample to obtain a measurement value for each of the at least two diagnostic markers; and (d) combining the measurements of the at least two diagnostic markers using a mathematical algorithm to obtain a TSH score.

[0202] Illustrative Implementation 40. The method of Illustrative Implementation 39, wherein the at least two diagnostic markers are TSH and a TSH variant characterized as R75G.

[0203] Illustrative Embodiment 41. The method of any one of Illustrative Embodiments 19-40, wherein the biological fluid sample is selected from blood, serum, plasma, saliva, sputum, mucus, nose, nasopharynx, anterior nose, oropharynx, trachea, bronchoalveolar and combinations thereof.

[0204] Illustrative Embodiment 42. The method of any one of Illustrative Embodiments 19-41, wherein the TSH score is used to support, predict, or replace thyroid testing.

[0205] Illustrative Implementation Scheme 43. The method of any one of Illustrative Implementation Schemes 19-42, wherein the mathematical algorithm is a discriminant function algorithm.

[0206] Illustrative Implementation Scheme 44. The method of Illustrative Implementation Scheme 43, wherein the discriminant function algorithm is a linear discriminant function algorithm.

[0207] Illustrative Implementation Scheme 45. The method of any one of Illustrative Implementation Schemes 19-44, wherein the TSH score is at least one factor in determining a treatment strategy for an individual.

[0208] Illustrative Implementation Scheme 46. The method of any one of Illustrative Implementation Schemes 19-45, wherein the TSH score is at least one factor for monitoring the efficacy of implementing a treatment strategy for an individual.

[0209] Illustrative Implementation Scheme 47. The method of any one of Illustrative Implementation Schemes 19-46, wherein the TSH score is at least one factor used to assess the degree of thyroid disease in an individual.

[0210] Illustrative Embodiment 48. A non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including any one of the methods in illustrative embodiments 19-47.

[0211] Illustrative Embodiment 49. A composition comprising: (a) two or more TSH biomarkers, wherein said one or more TSH biomarkers comprise: an anti-wild-type TSH antibody and an anti-TSH variant antibody. In some non-limiting embodiments, at least one anti-TSH biomarker reagent is artificial or synthetic.

[0212] 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.

[0213] Illustrative Embodiment 51. A kit for detecting thyroid diseases, the kit comprising: (a) two or more anti-TSH biomarker reagents, wherein the two or more anti-TSH biomarker reagents comprise: (i) at least one anti-TSH reagent, (ii) at least one anti-TSH variant reagent; and (b) instructions for use.

[0214] Illustrative Embodiment 52. A kit for detecting pituitary disorders or thyroid diseases, the kit comprising: (a) two or more anti-TSH biomarker reagents, wherein the two or more anti-TSH biomarker reagents are antibody reagents, and further comprising: (i) at least one anti-SEQ ID NO:1 reagent, (ii) at least one anti-SEQ ID NO:2 reagent; and (b) instructions for use.

[0215] Therefore, compositions, devices, and kits that fully satisfy the objectives and advantages set forth above, as well as methods of their production and use, have been provided in accordance with this disclosure. Although this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Accordingly, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

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

2. The kit according to claim 1, wherein the first label is selected from chemiluminescent compounds, phosphorescent compounds, fluorescent compounds, radioactive labels, biotin, and enzymes.

3. The kit according to claim 2, wherein the first label is acridine ester.

4. The kit according to claim 1, wherein the second label is selected from fluorescein, streptavidin, antibiotin antibody or its antigen-binding fragment, and digoxigenin.

5. The kit according to claim 4, wherein the second label is fluorescein, and wherein the third antibody or its antigen-binding fragment is an anti-fluorescein monoclonal antibody or its antigen-binding fragment.

6. The kit according to claim 5, wherein the fluorescein is fluorescein isothiocyanate (FITC).

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

8. The kit according to claim 1, wherein the TSH variant specifically bound to the third anti-TSH monoclonal antibody or its antigen-binding fragment is R75G.

9. The kit according to claim 1, wherein none of the first reagent, second reagent, third reagent and fourth reagent contains octylphenol polyoxyethylene ether.

10. The kit according to claim 1, wherein at least one of the first reagent, the second reagent, the third reagent, and the fourth reagent comprises nonylphenol polyoxyethylene ether.

11. A method for determining the total TSH concentration in a biological sample, the method comprising the following steps: (1) Combining (a)-(c) simultaneously or in whole or in part sequentially to form a mixture: (a) Biological samples; (b) A first reagent comprising a first label conjugated to a first anti-TSH monoclonal antibody or an antigen-binding fragment thereof; (c) A second reagent comprising a second label conjugated to a second anti-TSH monoclonal antibody or an antigen-binding fragment thereof; (d) A third reagent comprising a third anti-TSH monoclonal antibody or an antigen-binding fragment thereof, wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof specifically binds to at least one variant of TSH, and wherein the third anti-TSH monoclonal antibody or an antigen-binding fragment thereof is conjugated to a 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) The mixture was incubated under the following conditions: (b) and (c) were bound to TSH present in the biological sample to form a first sandwich complex; (b) and (d) were bound to a TSH variant present in the biological sample to form a second sandwich complex; and (e) was bound to an antibody with a second label to attach the first sandwich complex and the second sandwich complex to the solid support of (e). (3) Detect the signal generated by the first marker indirectly bound to the solid support of (e); and (4) Determine the total concentration of TSH present in the biological sample, wherein the concentration is proportional to the amount of signal generated by a first label indirectly bound to a solid support.

12. The method according to claim 11, wherein the biological sample is selected from blood, serum, plasma, saliva, sputum, mucus, nose, nasopharynx, anterior nose, oropharynx, trachea, 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, the chemiluminescence being quantified as relative light units (RLU).

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

15. The method of claim 11, wherein the solid support in (c) comprises paramagnetic particles.

16. The method of claim 11, wherein the third anti-TSH monoclonal antibody of (d) or the TSH variant specifically bound by its antigen-binding fragment is R75G.

17. The method of claim 11, wherein the method is performed in the absence of octylphenol polyoxyethylene ether.

18. The method of claim 11, wherein at least one of (b)-(e) comprises nonylphenol polyoxyethylene ether.

19. The method of claim 11, further defined as a method for determining the presence, severity, and / or susceptibility to thyroid disease or pituitary disorder in an individual.

20. The method of claim 11, wherein step (4) is further defined as comprising the following steps: (i) Measure the amounts of the first and second sandwich complexes formed to obtain a measurement of total TSH (total TSH + TSH variants) in the biological sample; and (ii) Use mathematical algorithms to obtain a TSH score based on the measurement of total TSH in the sample.

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