Compositions, kits, and systems for performing vitamin d assays and methods of producing and using same

Abstract

Compositions, reagents, kits, systems, and methods for performing Vitamin D immunoassays are disclosed. The compositions, reagents, kits, and systems rely on a first reagent that includes an anti-25-hydroxyvitamin D antibody or antigen-binding fragment thereof conjugated to a first label, and a preformed second reagent that includes a vitamin D analog conjugated to a second label along with a solid support conjugated to an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the antibody or antigen-binding fragment is bound to the second label so that the vitamin D analog is indirectly bound to the solid support during the manufacturing process.

Description

TITLECOMPOSITIONS, KITS, AND SYSTEMS FOR PERFORMING VITAMIN D ASSAYS AND METHODS OF PRODUCING AND USING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 729,686, filed December 9, 2024. The entire contents of the above- referenced patent application(s) are hereby expressly incorporated herein by reference.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via EFS-Web in computer readable form as an XML file. The Sequence Listing, created November 22, 2025, is named "2024P17488WO_SL.xml" and is 50,217 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference.BACKGROUND

[0004] Vitamin D is a steroid hormone involved in the intestinal absorption of calcium and the regulation of calcium homeostasis. Vitamin D is essential for the formation and maintenance of strong, healthy bones. Vitamin D deficiency can result from inadequate exposure to the sun, inadequate alimentary intake, decreased, absorption, abnormal metabolism, or vitamin D resistance. Recently, many chronic diseases such as cancer, high blood pressure, osteoporosis, and several autoimmune diseases have been linked to vitamin D deficiency.

[0005] Whether consumed or produced, both forms of vitamin D (D2and D3) are metabolized by the liver to 25-hydroxyvitamin D (also referred to herein as 25(OH)vitamin D), and then converted in the liver or kidney into 1,25-dihydroxyvitamin D. Vitamin D metabolites are bound to a carrier protein in the plasma and distributed throughout the body. It isgenerally accepted that the most reliable clinical indicator of vitamin D status is 25(OH)vitamin D because serum and plasma 25(OH)vitamin D levels reflect the body's storage levels of vitamin D, and 25(OH)vitamin D correlates with the clinical symptoms of vitamin D deficiency.

[0006] US Patent Nos. 8,785,603; 10,837,973; and 11,815,517 and US Patent Application Publication No. 2024 / 0094227 disclose antibodies to 25-hydroxyvitamin D2and D3, as well as methods of use thereof, including in the ADVIA CENTAUR® Vitamin D Total Assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The current ATELLICA® IM Vitamin D assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) is a competitive immunoassay that uses an anti-fluorescein mouse monoclonal antibody covalently bound to paramagnetic particles (PMP), an anti-25(OH)vitamin D mouse monoclonal antibody labeled with acridinium ester (AE), and a vitamin D analog labeled with fluorescein. An inverse relationship exists between the amount of vitamin D present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0007] However, type bias due to different matrices (serum, EDTA plasma, lithium heparin plasma, etc.) is a known potential issue in immunoassays. In particular, dose bias in the results of various Vitamin D immunoassays has been observed between blood collection tubes from different suppliers. For example, Chae et al. (Ann Lab Med (2024)44:611-613) disclosed that samples collected in Greiner Bio-One Vacuette serum-separator tubes (SSTs) exhibited higher values on the ATELLICA® IM Vitamin D Total assay compared to samples collected in the Becton Dickinson vacutainer SSTs (Becton Dickinson, Franklin Lakes, NJ), AB Medical V-Tubes with clot activator and gel (AB Medical, Seoul, Korea), and AB Medical VQ-tubes with clot activator and thrombin and gel. This bias would lead to incorrect classification of individuals as not deficient in vitamin D, despite having a 25(OH)D level of less than 20 ng / ml.

[0008] Therefore, there is a need in the art for new and improved reagents, kits, systems, and methods that overcome the bias resulting from variations in products utilized for sample collection.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0009] FIG. 1 illustrates a schematic of a prior art Vitamin D total assay.

[0010] FIG. 2 illustrates a schematic of one non-limiting embodiment of a vitamin D assay constructed in accordance with the present disclosure.

[0011] FIG. 3 illustrates a control comparison of assay panels of 25(OH)vitamin D2and 25(OH)vitamin D3when compared to LCMS (reference method), in which a traditional six-minute incubation period for the pretreated sample with the anti-vitamin D antibody AE conjugate. The Slope Difference (D2vs D3) for this control was 0.37 (i.e., almost a 40% variation).

[0012] FIG. 4 illustrates a comparison of assay panels of 25(OH)vitamin D2and 25(OH)vitamin D3when compared to LCMS, in which a one-minute incubation period for the pretreated sample with the anti-vitamin D antibody AE conjugate was utilized, along with a pretreatment reagent of 2x ANS. The Slope Difference (D2vs D3) for this experiment was -0.01.

[0013] FIG. 5 illustrates a comparison of assay panels of 25(OH)vitamin D2and 25(OH)vitamin D3when compared to LCMS, in which a one-minute incubation period for the pretreated sample with the anti-vitamin D antibody AE conjugate was utilized, along with a pretreatment reagent of 2.5x ANS. The Slope Difference (D2vs D3) for this experiment was 0.03.DETAILED DESCRIPTION

[0014] Before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components or steps or methodologies set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting in any way.

[0015] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0016] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by thoseof ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0017] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0018] All of the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods disclosed herein have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the present disclosure and / or appended claims.

[0019] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0020] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicatesotherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0021] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, includes ranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000, for example. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0022] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0023] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition " A or B" is satisfied by any of the following: A is true (orpresent) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0024] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0025] Throughout this application, the terms "about" and "approximately" are used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. That is, the terms "about" and "approximately" and variations thereof are intended to include not only the exact value qualified by the term, but to also include some slight deviations therefrom, such as deviations caused by measuring error, manufacturing tolerances, wear and tear on components or structures, settling or precipitation of cells or particles out of suspension or solution, chemical or biological degradation of solutions overtime, stress exerted on structures, and combinations thereof, for example. In particular, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, oreight percent, orseven percent, or six percent, orfive percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0026] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, unless otherwise noted, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may also include other elements not expressly listed or inherently present therein.

[0027] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, " A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0028] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0029] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0030] The term "isolated" as used herein means that a biological material, such as but not limited to a nucleic acid or protein, has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The same polynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being "isolated".

[0031] The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients, and the like.

[0032] The term "purified" as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of the natural material, for example but not by way of limitation, two, three, four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term "purified" as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.

[0033] The term "polynucleotide" or "oligonucleotide" as used herein will be understood to refer to a polymer of two or more nucleotides. Nucleotides, as used herein, will be understood to include deoxyribose nucleotides and / or ribose nucleotides, as well as artificial variants thereof. The term polynucleotide also includes single-stranded and double-stranded molecules.

[0034] As used herein, the terms "nucleic acid segment," "nucleic acid sequence," "nucleotide segment," "nucleotide sequence," " DNA sequence," and " DNA segment" are used interchangeably and refer to, for example, a synthetic DNA molecule or a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" or "isolated" nucleotide sequence as used herein refers to a DNA segment that is isolated away from, or purified free from, unrelated genomic DNA. Included within these terms are DNA segments and smaller fragments of such segments, and also recombinant vectors including, for example (but not by way of limitation), plasmids, cosmids, phage, viruses, and the like.

[0035] The term "polypeptide" as used herein refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. The term "polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids otherthan the 20 gene-encoded amino acids. The term "polypeptides" includes amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are wellknown in the art. Such modifications are well described in basic texts, monographs, and research literature. Modifications may occur anywhere in a polypeptide, includin the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural posttranslational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (See, for instance, Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., Analysis for Protein Modifications and Nonprotein Cofactors, Meth Enzymol (1990) 182:626-646 and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci (1992) 663:48-62).

[0036] The term "biomolecules" includes proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof.

[0037] The terms "express" and "produce" are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragmentthereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.

[0038] The term "specific binding partner" or "analyte-specific binder" will be understood to refer to any molecule capable of specifically associating with a target analyte. For example, but not by way of limitation, the binder / binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), any fragments thereof, and any combinations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte.

[0039] The term "antibody" is used in the broadest sense, and specifically (but not by way of limitation) covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired biological activity of analyte binding. Thus, the term "antibody" or "antibody peptide(s)" refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(a b')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, e.g., Hudson et al. (Nature Med. (2003) 9:129-134). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).

[0040] The term "antigen binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigenbinding fragment" of an antibody include but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments areobtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[0041] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0042] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0043] The terms " CDR," and its plural " CDRs," refer to a complementarity determining region (CDR) of an antibody or antibody fragment, which determine the binding character of an antibody or antibody fragment. In most instances, three CDRs are present in a light chain variable region (CDRL1, CDRL2 and CDRL3) and three CDRs are present in a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. Among the various CDRs, the CDR3 sequences, and particularly CDRH3, are the most diverse and therefore have the strongest contribution to antibody specificity. There are at least two techniques for determining CDRs: (1) an approach based on crossspecies sequence variability (i.e., Kabat et al.. Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety).

[0044] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a "conformational epitope"), as well as specific charge characteristics.

[0045] An epitope is defined as "the same" as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise epitopes that are the same. In certainembodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.

[0046] An antibody "specifically binds" an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody comprises an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, the antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope or closely related epitopes. In certain instances, for example, homologous proteins from different species may comprise the same epitope. In certain embodiments, an antibody specifically binds to an antigen with a dissociation constant of no greater than 10-6M, 10-7M, 10-8M or 10-9M. When an antibody specifically binds to a receptor or ligand (i.e., counterreceptor), it may substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when an excess of antibody reduces the quantity of receptor bound to ligand by at least about 20%, 40%, 60% or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0047] An "isolated" antibody is one which has been separated and / or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, alternatively, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the environment in which the antibody is produced will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In addition, the "isolated antibody" is substantially free of other antibodies havingdifferent antigenic specificities. An isolated antibody may, however, have some crossreactivity to other, related antigens.

[0048] The term "antibody mutant" refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of eitherthe heavy or light chain variable domain of the antibody, such as at least 80%, or at least 85%, or at least 90%, or at least 95%.

[0049] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that in one method of production they may be synthesized by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, in one embodiment, the monoclonal antibodies produced in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0050] The monoclonal antibodies utilized in accordance with the present disclosure may be produced by any methodology known in the art including, but not limited to, a result of a deliberate immunization protocol; a result of an immune response that results in the production of antibodies naturally in the course of a disease or cancer; phage-derived antibodies; and the like. In addition to the hybridoma production method listed above, the monoclonal antibodies of the present disclosure may be produced by other various methods such as, but not limited to, recombinant DNA methods (see, e.g., U. S. Pat. No. 4,816,567); isolation of antibody fragments from a phage display library (see, e.g., Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); as well as various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.)). Further, many monoclonal antibodies that may be utilized in the conjugates and methods disclosed or otherwise contemplated herein are widely commercially available, and therefore no further description thereof is deemed necessary.

[0051] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Generally, a substantially pure composition will comprise more than about 50% percent of all macromolecular species present in the composition, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0052] An "analyte" is a molecule that is capable of being recognized by an analytespecific binding partner, such as (but not limited to) an antibody. An analyte comprises at least one antigenic determinant or "epitope," which is the region of the analyte which binds to the analyte-specific binding partner (i.e., antibody).

[0053] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another as well as indirect association / binding of two moieties to one another. Non-limiting examples of associations / couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.

[0054] Circuitry, as used herein, may be analog and / or digital components, or one or more suitably programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. Also, a "processing component" may perform one or more functions. The term "processing component," may include hardware, such as a processor (e.g., microprocessor), an application specific integrated circuit (ASIC), field programmable gate array (FPGA), a combination of hardware and software, and / or the like.

[0055] Software may include one or more computer readable instructions that when executed by one or more processing components cause the processing component toperform a specified function. It should be understood that the algorithms described herein may be stored on one or more non-transitory memory. Exemplary non-transitory memory may include random access memory, read only memory, flash memory, and / or the like. Such non-transitory memory may be electrically based, optically based, and / or the like.

[0056] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value.

[0057] It is to be further understood that, as used herein, the term "user" includes but is not limited to a human being, and may comprise, a computer, a server, a website, a processor, a network interface, a human, a user terminal, a virtual computer, combinations thereof, and the like, for example.

[0058] The term "calibration parameters" as used herein refers to a collection of data points or one or more functions used to derive a collection of data points that correlates the signals from the sensor to known analyte concentrations. The calibration parameters can be derived by a calibration algorithm, such as a linear algorithm, a spline-based algorithm, exponential algorithm, a least squares algorithm, a logarithmic algorithm, or the like that is configured to fit a function to at least two calibration points.

[0059] The term "calibration logic" as used herein refers to the program logic used by a processing component to interpret data measured by one or more electrodes. In particular, the term "calibration logic" is the program logic used by a processing component to interpret data from an electrochemical sensor having at least a working electrode and a reference electrode.

[0060] The term "sample" as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood orany portion thereof (i.e., plasma or serum), urine, saliva, sputum (such as, but not limited to, bronchoalveolar lavage sputum), cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and the like, as well as any combinations thereof.

[0061] Turning now to the inventive concepts, certain non-limiting embodiments of the present disclosure are directed to compositions, reagents, kits, and systems designed for use in methods of performing Vitamin D immunoassays, as well as methods of producing and using same. The immunoassay has a competitive format that uses a first reagent comprising an anti-25(OH) vitamin D antibody or antigen-binding fragment thereof conjugated to a first label, and a preformed second reagent that comprises a conjugate of a vitamin D analog and a second label along with a solid support coated with an antibody or other binding partner that specifically binds to the second label, wherein the antibody binds to the second label of the conjugate so that the vitamin D analog is bound to the solid support during manufacturing of the reagents. In the immunoassay, the antibody (or antigen-binding fragment thereof) of the first reagent specifically binds to the vitamin D analog of the second reagent or to vitamin D present in a biological sample. An inverse relationship exists between the amount of vitamin D present in the biological sample and the amount of signal generated by the first label when it is indirectly bound to the solid support that is detected by the system.

[0062] As stated herein above, dose bias in the results of various Vitamin D immunoassays has been observed between blood collection tubes from different suppliers, and this bias would lead to incorrect classification of individuals as not deficient in vitamin D, despite having a 25(OH)D level of less than 20 ng / ml. By preforming the second reagent during the manufacturing process (as opposed to allowing the conjugate and coated solid support to bind to one another in the cuvette during the reaction with the sample), less tube type bias was observed. In addition, use of the preformed second reagent also has the benefits of being less sensitive to ambient light exposure along with less assay interference in patients exposed to treatments containing of the binding partners attached to the vitamin D analog or coated on the solid support (such as, but not limited to, fluorescein treatment, biotin supplementation, etc.).

[0063] Certain non-limiting embodiments of the present disclosure include a kit for performing an immunoassay for Vitamin D. The kit includes a first reagent comprising a firstlabel conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof. The kit also includes a preformed second reagent that includes a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support such that the vitamin D analog is indirectly bound to the solid support during the manufacturing process. In addition, the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent.

[0064] In certain particular (but non-limiting) embodiments, the kits may optionally include a third, displacement reagent comprising at least one releasing agent. In a particular (but non-limiting) embodiment, the third reagent further comprises polyvinylpyrrolidone (PVP).

[0065] Certain non-limiting embodiments of the present disclosure are directed to systems for performing an immunoassay for Vitamin D, wherein the system comprises any of the kits disclosed or otherwise contemplated herein in combination with a sample collection tube (such as, but not limited to, a serum separator tube).

[0066] In certain particular (but non-limiting) embodiments, the sample collection tube utilized in the kits, systems, and methods of the present disclosure includes PVP which can contribute to a dose bias in the results of various Vitamin D immunoassays of the prior art. Therefore, the polyvinylpyrrolidone (PVP) present in the third / displacement reagent of the kits of the present disclosure can assist in eliminating tube type bias by being provided at an amount sufficient to saturate out the PVP in the sample collection tube to thereby eliminate dose bias.

[0067] The kits and systems of the present disclosure may further contain one or more additional reagents or devices for use in the methods of the present disclosure. For example (but not by way of limitation), the kit or system may further include at least one additional reagent for initiating detection of the first label. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary.

[0068] Certain non-limiting embodiments of the present disclosure include a method of determining a concentration of vitamin D in a biological sample. In the method, step (1)includes combining, either simultaneously or wholly or partially sequentially, the following components (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support to provide a preformed reagent prior to reaction with (a) and (b). In addition, the antibody or antigen-binding fragment thereof of the first reagent of (b) specifically binds to the vitamin D analog of the preformed second reagent of (c). In step (2), the mixture is incubated under conditions whereby (b) binds to vitamin D present in the biological sample. In step (3), a signal generated by the first label on the solid support is detected. In step (4), a concentration of vitamin D present in the biological sample is determined, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0069] Certain non-limiting embodiments of the present disclosure include method of determining a concentration of vitamin D in a biological sample. In the method, step (1) includes combining, either simultaneously or wholly or partially sequentially, the following components (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support to provide a preformed reagent prior to reaction with (a) and (b). In addition, the antibody or antigen-binding fragment thereof of the first reagent of (b) specifically binds to the vitamin D analog of the preformed second reagent of (c). In step (2), the mixture is incubated under conditions whereby (b) binds to vitamin D present in the biological sample. In step (3), a signal generated by the first label on the solid support is detected. In step (4), a concentration of vitamin D present in the biological sample is determined, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0070] Certain non-limiting embodiments of the present disclosure include a method of detecting vitamin D deficiency in a subject. In the method, step (1) includes combining, either simultaneously or wholly or partially sequentially, components (a)-(c) to form a mixture: (a) a biological sample derived from the subject; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support to provide a preformed reagent prior to reaction with (a) and (b). In addition, the antibody or antigen-binding fragment thereof of the first reagent of (b) specifically binds to the vitamin D analog of the preformed second reagent of (c). In step (2), the mixture is incubated under conditions whereby (b) binds to vitamin D present in the biological sample. In step (3), a signal generated by the first label on the solid support is detected. In step (4), a concentration of vitamin D present in the biological sample is determined, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0071] In certain particular (but non-limiting) embodiments, a decrease between the level of vitamin D in the biological sample relative to the level in a normal control or a threshold level of 30 ng / mL is indicative of a vitamin D deficiency in said subject.

[0072] Certain non-limiting embodiments of the present disclosure include a method for treating a subject suspected of having a vitamin D deficiency. In the method step (1) includes combining, either simultaneously or wholly or partially sequentially, components (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support to provide a preformed reagent prior to reaction with (a) and (b). In addition, the antibody or antigenbinding fragment thereof of the first reagent of (b) specifically binds to the vitamin D analog of the preformed second reagent of (c). In step (2), the mixture is incubated under conditionswhereby (b) binds to vitamin D present in the biological sample. In step (3), a signal generated by the first label on the solid support is detected. In step (4), a concentration of vitamin D present in the biological sample is determined, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support. In step (5), if a decrease between the level of vitamin D in the biological sample relative to the level in a normal control or a threshold level of 30 ng / mL is determined, a treatment for vitamin D deficiency is administered to said subject.

[0073] Certain non-limiting embodiments of the present disclosure include a method for monitoring progression of vitamin D deficiency in a subject in need thereof. In the method, step (i) involves performing steps (1)-(4) of any of previously disclosed methods using a first biological sample collected at a first time point, while step (ii) includes performing steps (1)-(4) of any of the previously disclosed methods using a second biological sample collected at a second time point after the first time point. A decrease between the level of vitamin D present in the first biological sample and the level of vitamin D present in the second biological sample is indicative of the progression of a vitamin D deficiency in said subject; little or no change between the levels of vitamin D in the first and second biological samples is indicative of stabilization of a vitamin D deficiency in the subject; and an increase between the level of vitamin D in the first biological sample and the level of vitamin D in the second biological sample is indicative of regression of a vitamin D deficiency in the subject.

[0074] Certain non-limiting embodiments of the present disclosure include a method for monitoring treatment of vitamin D deficiency in a subject in need thereof. In the method, step (i) includes performing steps (1)-(4) of any of previously described methods using a first biological sample collected at a first time point, while step (ii) includes performing steps (1)-(4) of any of the previously described methods using a second biological sample collected at a second time point after the first time point and following administration to the subject of a treatment for vitamin D deficiency. An increase in or stabilization of the level of vitamin D in the second biological sample relative to the level of vitamin D in the first biological sample is indicative of efficacy of the treatment of the vitamin D deficiency in said subject, and a decrease in the level of vitamin D in the second biological sample relative to the level of vitamin D in the first biological sample is indicative of inefficacy of the treatment of the vitamin D deficiency in said subject.

[0075] The kits, systems, and methods of the present disclosure may be utilized with any biological samples known in the art or otherwise disclosed herein. In a particular (but nonlimiting) embodiment, the biological sample is at least one of blood, serum, or plasma.

[0076] In addition, any of the methods disclosed or otherwise contemplated herein may include one or more additional steps. Non-limiting examples of additional steps that may be utilized include performing a separation of the biological sample priorto step (1) using at least one sample collection tube; performing a wash step prior to step (3); repeating one or more steps; and the like, as well as any combinations thereof. When one or more additional steps are present, the kits and systems of the present disclosure may further include one or more reagents utilized in such additional steps.

[0077] In certain particular (but non-limiting) embodiments, step (1) of any of the methods disclosed or otherwise contemplated herein further includes adding a third reagent (d) simultaneously or wholly or partially sequentially with (a)-(c). Reagent (d) is a displacement reagent that comprises at least one releasing agent. In a particular (but nonlimiting) embodiment, reagent (d) also comprises PVP.

[0078] The components (a)-(c) (and component (d), when present) may be combined simultaneously or in any order in any of the methods disclosed or otherwise contemplated herein. In one particular (but non-limiting) embodiment, the components are combined simultaneously. In another particular (but non-limiting) embodiment, components (a) and (d) are combined prior to addition of components (b)-(c).

[0079] Certain particular (but non-limiting) embodiments of the present disclosure include any of the methods disclosed or otherwise contemplated herein method of determining a concentration of vitamin D in a biological sample, in which the reagents are added in a specific order and include one or more specific incubation steps. For example (but not by way of limitation), the biological sample may first be combined with the displacement reagent that comprises at least one releasing agent, and then the reagent comprising the first label conjugated to the anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof is added thereto, and the resultant mixture is incubated for a specific period of time and under conditions whereby the anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof binds to vitamin D present in the biological sample. Then the preformed reagent is added following this incubation step.

[0080] The pretreated sample (i.e., the sample that has been incubated with the displacement reagent containing the releasing agent) is incubated with the reagent comprising the first label conjugated to the anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof for a specific period of time such as, but not limited to, about 90 seconds, about 80 seconds, about 75 seconds, about 70 seconds, about 65 seconds, about 60 seconds, about 55 seconds, about 50 seconds, about 45 seconds, about 40 seconds, about 35 seconds, about 30 seconds, or less, as well as a range formed from two of any of the above values (such as, but not limited to, a range of from about 30 seconds to about 90 seconds, etc.).

[0081] In certain particular (but non-limiting) embodiments, the kits, systems, and methods of the present disclosure may detect 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

[0082] Any anti-25-hydroxyvitamin D monoclonal antibodies or antigen-binding fragments thereof known in the art or otherwise contemplated herein may be utilized in the kits, systems, and methods in accordance with the present disclosure. Non-limiting examples of monoclonal antibodies or antigen-binding fragments thereof are disclosed in US Patent Nos. 8,785,603; 10,837, 973; and 11,815,517 and US Patent Application Publication No.2024 / 0094227, along with other commercially available antibodies known in the art or otherwise contemplated herein. The antibody sequences for the antibodies disclosed in the '603, '973, and '517 patents and the '227 patent application publication are incorporated herein, as shown in Table 1.TABLE 110H9 Antibody Segment Amino Acid SEQ ID NO: DNA SEQ ID NO:Lc CDR1 26 18 Lc CDR2 27 19 Lc CDR3 28 20 Lc FWR1 29 21 Lc FWR2 30 22 Lc FWR3 31 23 Lc FWR4 25 17 Lc variable domain 32 24Hc CDR1 10 2 Hc CDR2 11 310H9 Antibody Segment Amino Acid SEQ ID NO: DNA SEQ ID NO:Hc CDR3 12 4 He FWR1 13 5 He FWR2 14 6 He FWR3 15 7 He FWR4 9 1 He variable domain 16 8

[0083] In some non-limiting embodiments, the anti-25(OH) vitamin D antibodies or antigen-binding fragments may include one or more of a heavy chain CDR1 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 10; a heavy chain CDR2 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 11; a heavy chain CDR3 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 12; a light chain CDR1 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 26; a light chain CDR2 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 27; and / or a light chain CDR3 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 28.

[0084] In some non-limiting embodiments, the anti-25(OH) vitamin D antibodies or antigen-binding fragments may include one or more of a heavy chain FWR1 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 13; a heavy chain FWR2 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 14; a heavy chain FWR3 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 15; a light chain FWR1 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 29; a light chain FWR2 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 30; and / or a light chain FWR3 amino acid sequence substantially the same as, or identical to, SEQ ID NO: 31.

[0085] In some non-limiting embodiments, the anti-25(OH) vitamin D antibodies or antigen-binding fragments may include one or more of a heavy chain CDR1 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 2; a heavy chain CDR2 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 3; a heavy chain CDR3 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 4; a light chain CDR1 amino acid sequence encoded by a polynucleotidesequence substantially the same as, or identical to, SEQ ID NO: 18; a light chain CDR2 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 19; and / or a light chain CDR3 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 20.

[0086] In some non-limiting embodiments, the anti-25(OH) vitamin D antibodies or antigen-binding fragments may include one or more of a heavy chain FWR1 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 5; a heavy chain FWR2 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 6; a heavy chain FWR3 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 7; a light chain FWR1 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 21; a light chain FWR2 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 22; and / or a light chain FWR3 amino acid sequence encoded by a polynucleotide sequence substantially the same as, or identical to, SEQ ID NO: 23.

[0087] The phrase "substantially the same" with respect to the above nucleic acid or amino acid sequences, means at least 65% identity between two or more sequences, such as (but not limited to) at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% or greater identity between two or more sequences. Such identity may be determined using mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87:2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-7).

[0088] Because of the natural sequence variation likely to exist among heavy and light chains and the genes encoding them, one would expect to find some level of variation within the amino acid sequences or the genes encoding the antibodies or antigen-binding fragments described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). Such an expectation is due in part to the degeneracy of the genetic code, as well as to the evolutionary success of conservative amino acid sequence variations, which do not appreciably alterthe nature ofthe encoded protein. Accordingly, some embodiments include antibodies or antigen-binding fragments having 90%, 95%, 96%, 97%, 98%, or 99% homology to the antibodies or antigen-binding fragments herein. Other embodiments includeantibodies that preferentially bind a vitamin D derivative, such as 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenic molecule or compound, or antigen-binding fragments of such antibodies, that have framework, scaffold, or other non-binding regions that do not share significant homology with the antibodies and antigen-binding fragments described herein, but do incorporate one or more CDRs or other sequences needed to confer binding that are 90%, 95%, 96%, 97%, 98%, or 99% homologous to such sequences described herein.

[0089] The antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or binding preference) of the described antibodies or antigen-binding fragments. The skilled person may produce variants having single or multiple amino acid substitutions, deletions, or additions. These variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the person having ordinary skill in the art.

[0090] Any of the antibodies or antigen-binding fragments described or otherwise herein may embody several antibody isotypes, such as (but not limited to) IgM, IgD, IgG, IgA, and IgE. Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotypeto another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0091] Any of the antibodies or antigen-binding fragments described or otherwise contemplated herein have binding affinities (in M) for a vitamin D derivative, such as 25- hydroxyvitamin D2 and / or 25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenic molecule or compound that include a dissociation constant ( D) of less than IxlO"2. In some embodi ents, the Kois less than IxlO"3. In other embodiments, the Ko is less than IxlO"4. In some embodiments, the Kois less than 1x10"-’. In still other embodiments, the KD I’S less than IxlO’6, 2xl0’6, 3xl0’6, 4xl0’6, 5xl0"6, 6xl0’6, 7xl0’6, 8xl0“6, or 9X10"6. In other embodiments, the KD is less than IxlO’7, 2xl0’7, or 3x10“7, 2xl0’7, 3xl0“7, 4xl0“7, 5xl0“7, 6xl0“7, 7x10“7, 8xl0“7, or 9xl0“7. In other embodiments, the KD is less than IxlO’8, 2xl0“8, 3xl0’8, 4xl0"8, 5xl0“s, 6xl0“s, 7xl0“8, 8xl0’8, or 9xl0“8. In other ernbodiments, the Kpis less than IxlO’9, 2.xl0'9, 3xl0’9, 4xlQ“9, 5xl0’9, 6xl0’9, 7xl0“9, 8xl0’9, or 9xl0“9. In other embodiments, the KDis less than IxlO"10, 2xlO”10, 3xl0“10, 2xlO“10, 3xlO’10, 4xlO”10, 5xl0”:!°, 6x10 ™ 7x10’™, 8x10 ™, or 9x10”™. In still other embodiments, the Ko is less than IxlO’11, 2. X1011, 3X10’11, 4xl0“u, SxlO’11, 6xl0"n, 7X10’11, 8xl0”n, or 9x10"n. In some embodiments, the Kois less than IxlO’11. In other embodiments, the Kois less than IxlO13. In other embodiments, the Kois less than 1x1014In still other embodiments, the KD is less than IxlO’15.

[0092] The antibodies or antigen-binding fragments described herein, in some embodiments, have equimolar recognition of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0093] In addition to the anti-25-hydroxyvitamin D antibodies for which the sequences thereof are provided herein, other anti-25-hydroxyvitamin D monoclonal antibodies or antigen-binding fragments thereof are widely known in the art and commercially available and thus can be utilized in the kits, systems, and methods in accordance with the present disclosure. Non-limiting examples of commercial sources that supply anti-25-hydroxyvitamin D monoclonal antibodies or antigen-binding fragments thereof include Antibod ies-On line Inc. (Limerick, PA); Creative Biolabs, Inc. (Shirley, NY); Creative Diagnostics (Shirley, NY); Immunodiagnostik AG (Bensheim, Germany); My BioSource, Inc. (San Diego, CA); US Biological Life Sciences (Salem, MA); and many others. However, this list is not inclusive, and there are many additional commercial sources of anti-25(OH) vitamin D antibodies that can be utilizedin accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be abie to identify and select a variety of anti-25(OH) vitamin D antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-25(OH) vitamin D antibodies or the characteristics thereof is deemed necessary.

[0094] Any of the antibodies or antigen-binding fragments described or otherwise contemplated herein may be labeled or otherwise conjugated to various chemical or biomolecule moieties for use in the diagnostic applications disclosed herein. The moieties may be detectable labels, for example, chemiluminescent labels (e.g., acridinium esters and sulfonamides, luminol and isoluminol), phosphorescent labels, fluorescent labels (e.g., FITC), electrochemiluminescent labels (e.g., ruthenium (II) chelates), cloned enzyme donors, photosensitizer particles or chemiluminescer particles for luminescent oxygen channeling immunoassays (LOCI), lanthanide chelates for time-resolved fluorescence immunoassays (TR-FIA), radiolabels, biotin, digoxigenin, enzymes and the like, for example, radionuclides, such as, but not. limited to, tritium, carbon-14, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-ill, and fissionable nuclides such as boron-10 or an actinide. In some embodiments, enzymes may be conjugated to the described antibodies or antigen-binding proteins for the purposes of detecting bound antibody in a sample. Such enzyme conjugates include, but are not limited to, alkaline phosphatase (AP), horseradish peroxidase, beta¬ galactosidase and glucose-6-phosphate dehydrogenase (G6PDH). Other enzymes used to determine antibody binding in solution-based immunoassays would be understood by those skilled in the art to be suitable for use as a conjugate for the antibodies and antigen-binding fragments described herein. In addition, compounds such as acridinium esters may also be conjugated to the provided antibodies and antigen-binding fragments to allow for detection in an immunoassay.

[0095] In certain particular (but non-limiting) embodiments, the first label conjugated to the anti-25(OH) vitamin D antibody or antigen binding fragment thereof is a detectable label. For example (but not by way of limitation), the first label can be a chemiluminescent compound (e.g., an acridinium ester compound), a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and / or an enzyme. These exemplary labels can usually only be detected when excited by methods that include, but are not limited to, addition of1different chemicals, stimulation by light, or exposure to substrate or other compounds. When using an acridinium ester compound, chemiluminescence is triggered by peroxide and acid / base, resulting in a flash that can be read by appropriate instrumentation. In addition, an optional wash step may be used before initiating detectability of the detectable label.

[0096] In a particular (but non-limiting) embodiment, the first label is acridinium ester. In this instance, step (3) of any of the methods disclosed or otherwise contemplated herein may comprise adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0097] The second label conjugated to the vitamin D analog may be any molecule for which an antibody or other specific binding partner is available that can be conjugated to the solid support, so long as the second label and the antibody / binding partner do not otherwise interfere with the assay being performed. Non-limiting examples of second labels and antibody / specific binding partner combinations that can be utilized in the kits, systems, and methods in accordance with the present disclosure include fluorescein (e.g., fluorescein isothiocyanate (FITC)) for binding to anti-fluorescein antibody or fragment thereof; biotin for binding to avidin, streptavidin, or anti-biotin antibody; digoxigenin for binding to anti-digoxigenin antibody; other hapten and binding partner combinations; and the like. These types of label and antibody / specific binding partner combinations are well known in the art and widely available commercially. Thus, no further description thereof is deemed necessary.

[0098] Any vitamin D analogs capable of specifically binding to the anti-25(OH) vitamin D monoclonal antibody orantigen-bindingfragment thereof may be utilized in the kits, systems, and methods in accordance with the present disclosure and conjugated to the second label. Non-limiting examples of vitamin D analogs that may be utilized in accordance with the present disclosure include 24R-Calcipotriol, Lythgoe diol 4-TBDMS ether, 25-Hydroxyvitamin D3 3-Hemisuccinate, Calcitriol-d6, Lythgoe diol, 25-OH vitamin D3-DPEG4-Biotin, 25-OH vitamin Ds-LC-Biotin, vitamin D-C22 acid, and the like. Particular (but non-limiting) examples of vitamin D analogs may be based on the use of a vitamin D 22 carbon derivative (vitamin D-C22), which includes a C22 carboxy group when unconjugated. In s particular (but nonlimiting) embodiment, the vitamin D analog may be vitamin D-C22.

[0099] Any solid supports known in the art and capable of functioning in accordance with the kits, systems, and methods described herein may be utilized in accordance with the present disclosure. Non-limiting examples of solid phase supports that may be utilized includeparamagnetic particles (PMPs); cross-linked dextran available under the trademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N. J.); agarose; polystyrene beads; polyvinyl chloride; polystyrene; cross-linked polyacrylamide; nitrocellulose- or nylon-based webs such as sheets, strips, or paddles; or tubes, plates, or wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. When using paramagnetic particles, some source of a magnetic field may be used to retain the particles and molecules bound directly or indirectly to the particles during an optional wash step. The molecules may be bound covalently, by salt-bridges, hydrogen bonding, or another type of bond.

[0100] The optional third reagent of the kits, systems, and methods of the present disclosure is a displacement buffer that comprises at least one releasing agent. Non-limiting examples of releasing agents that may be utilized in accordance with the present disclosure include 8-anilino-1-naphthalene sulfonate (ANS), dithiothreitol (DTT), TRIS (2-carboxyethyl) phosphine (TCEP), sodium hydroxide (NaOH), and the like, as well as any combinations thereof. In particular (but non-limiting) embodiments, when ANS is present, the ANS may be in the form of ANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt, ANS hemimagnesium salt, ANS ammonium salt, or the like). The optional third reagent / displacement buffer may further contain one or more additional ingredients, such as (but not limited to) PVP, ethylene glycol, EDTA, citrate, methanol, and / or sodium azide. In a particular (but non-limiting) embodiment, the optional third reagent / displacement buffer comprises ANS, ethylene glycol, methanol, and PVP.

[0101] The compositions / reagents of the kits or systems may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and / or single aliquot form within the kit or system. Alternatively, in a particular (but nonlimiting) embodiment, one or more of the reagents may be disposed in the kit or system in the form of a single aliquot lyophilized reagent. The use of dried reagents in kits / microfluidics devices is described in detail in US Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0102] Also, the compositions / reagents present in the kits or systems may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. In addition, the kit or system may include a device (such as, but notlimited to, a microfluidics device or cartridge for loading onboard of a clinical instrument) in which one or more of the compositions / reagents are disposed.

[0103] The relative amounts of the various compositions / reagents in the kits or systems can vary widely to provide for concentrations of the compositions / reagents that substantially optimize the reactions that need to occur during the methods and further to optimize substantially the sensitivity and selectivity of an assay. Under appropriate circumstances, one or more of the compositions / reagents in the kit or system can be provided as a dry powder, such as a lyophilized powder, and the kit or system may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these compositions. Quality control and / or calibration reagent(s) may also be included with the kit or system. In addition, the kit or system can further include a set of written instructions (or access to electronic instructions) explaining how to use the kit or system. A kit or system of this nature can be used in any of the methods described or otherwise contemplated herein.EXAMPLES

[0104] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.Example 1

[0105] FIG. 1 illustrates a prior art Vitamin D total assay, such as is described in US Patent Nos. 8,785,603; 10,837,973; and 11,815,517 and US Patent Application Publication No.2024 / 0094227, and as utilized in currently commercially available ADVIA CENTAUR® Vitamin D Total Assays and ATELLICA® IM Vitamin D assays (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The illustrated assay utilizes an anti-25(OH)vitamin D mouse monoclonal antibody labeled with acridinium ester (AE) (indicated in the second section of FIG. 1 and labeled as " Lite Reagent"), an anti-fluorescein mouse monoclonal antibody covalently bound to paramagnetic particles (PMP) (indicated in the third section of FIG. 1 and labeled as " PMP a-FITC"), a vitamin D analog labeled with fluorescein (indicated in the third section of FIG. 1and labeled as " C22 VD" and " FL"), and a fourth, displacement reagent (indicated in the first section of FIG. 1 and labeled as " Releasing Reagent"). An inverse relationship exists between the amount of vitamin D present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0106] As stated herein above, dose bias in the results of various Vitamin D immunoassays has been observed between blood collection tubes from different suppliers, and this bias would lead to incorrect classification of individuals as not deficient in vitamin D, despite having a 25(OH)D level of less than 20 ng / ml. It has surprisingly been found herein that preforming a second reagent comprising the vitamin D analog indirectly bound to a solid support during the manufacturing process (as opposed to allowing the vitamin D analogcontaining conjugate and the coated solid support to bind to one another in the cuvette during the reaction with the sample), less tube type bias was observed. In addition, use of the preformed second reagent also has the benefits of being less sensitive to ambient light exposure along with less assay interference in patients exposed to treatments containing of the binding partners attached to the vitamin D analog or coated on the solid support (such as, but not limited to, fluorescein treatment, biotin supplementation, etc.).

[0107] FIG. 2 illustrates one non-limiting embodiment of an immunoassay architecture constructed in accordance with the present disclosure. The immunoassay utilizes a first reagent that includes an anti-25(OH)vitamin D mouse monoclonal antibody labeled with acridinium ester (AE) (indicated in the second section of FIG. 2 and labeled as " Lite Reagent"), and a preformed second reagent comprising an anti-fluorescein mouse monoclonal antibody covalently bound to paramagnetic particles (PMP) (indicated in the third section of FIG. 2 and labeled as " PMP a-FITC"), wherein the anti-fluorescein antibody is bound to a conjugate of fluorescein conjugated to a vitamin D analog (indicated in the third section of FIG. 2 as being prebound to the coated PMP and labeled as " C22 VD" and " FL"). The assay may optionally include a third, displacement reagent (indicated in the first section of FIG. 2 and labeled as " Releasing Reagent"). An inverse relationship exists between the amount of vitamin D present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0108] In addition to the benefits mentioned above, the novel immunoassays of the present disclosure can be completed in almost half of the time required to complete the prior art immunoassay. FIG. 1 indicates that the prior art immunoassay requires 18.25 minutes tocomplete, whereas the novel immunoassays of the present disclosure can be completed in less than 10 minutes (FIG. 2 indicates a total of 9.8 minutes).

[0109] The primary function of the optional third / displacement reagent comprising the at least one releasing agent is to release vitamin D from its binding protein present in a biological sample. This reaction is typically added to the cuvette after sample addition. Once vitamin D is released from the binding protein, it is available to bind to the antibody utilized in the assay. In the present disclosure, polyvinylpyrrolidone (PVP) may also be present in the third / displacement reagent. PVP is used to further assist in eliminating tube type bias and is provided at an amount sufficient to saturate out the PVP in certain tube types to thereby eliminate dose bias between tube types.

[0110] Table 2 illustrates the dose bias observed between certain tube types in the commercially available ATELLICA® IM Vitamin D assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). Vitamin D assay values for samples from various subjects were compared when collected in various BD tubes (Becton Dickinson, Franklin Lakes, NJ) versus various Greiner tubes (Greiner Bio-One North America Inc., Monroe, NC). The dose values represented in Table 2 are in ng / mL. As can be seen, a 22% average bias was observed for Greiner SST tubes, and a 12% average bias was observed for Greiner SST P tubes. No bias was observed for the Greiner LiHep tubes.TABLE 2Donor 1 2 3 4 5 6 7 8 9 10 BD LiHep 18.51 74.03 27.41 22.62 36.09 34.50 19.37 22.51 36.65 23.45 BD LiHep Gel 21.54 75.68 29.97 24.42 36.66 33.81 20.96 22.40 40.70 23.56 BD Red Top 19.55 69.97 26.92 20.61 33.02 31.22 17.54 20.42 34.18 22.92 BD SST 18.91 75.66 27.78 21.78 36.93 29.78 18.91 21.92 34.60 23.60 Greiner LiHep Gel 19.96 77.14 30.00 22.52 34.68 34.17 19.72 22.39 37.29 24.01 Greiner SST 21.53 80.87 33.71 27.40 41.68 38.04 25.45 31.86 38.64 27.33 Greiner SST P 20.80 79.27 31.14 26.01 38.70 35.85 22.49 24.17 38.19 24.75SST Tubes % Dose Difference Avg Greiner SST vs 14% 7% 21% 26% 13% 28% 35% 45% 12% 16% 22% BD SSTGreiner SST P vs 10% 5% 12% 19% 5% 20% 19% 10% 10% 5% 12% BD SSTLiHep Tubes % Dose DifferenceGreiner LiHep Gel -7% 2% 0% -8% -5% 1% -6% 0% 12% 2% -1%vs BD LiHep GelGreiner LiHep Gel 8% 4% 9% 0% -4% -1% 2% -1% 2% 2% 2%vs BD LiHep

[0111] A similar analysis was conducted using different formulations of two components of the Vitamin D assay, with the amount of ANS in the displacement reagent doubled (2x ANS) and the amount of AE-labeled antibody doubled (2xAE), and the results are shown in Table 3. The dose values represented in Table 3 are in ng / mL. As can be seen, a 64% average bias was observed for Greiner SST tubes, and a 40% average bias was observed for Geiner SST P tubes. An average bias of 6% or less was observed for Greiner LiHep.TABLE 3Donor 1 2 3 4 5 6 7 8 9 10 BD LiHep 13.01 64.30 21.67 16.70 25.53 26.41 12.47 14.77 25.70 15.23 BD LiHep Gel 14.82 64.23 22.27 16.59 27.24 25.17 13.93 14.44 27.04 15.56 BD Red Top 13.12 56.81 19.45 15.23 24.59 23.56 10.79 15.76 23.15 14.21 BD SST 17.51 71.54 24.13 20.09 30.29 30.62 17.05 19.61 30.07 19.88 Greiner LiHep Gel 13.32 62.59 22.38 16.90 28.44 26.76 13.27 16.79 28.97 16.04 Greiner SST 27.32 108.41 43.36 39.61 48.72 50.26 28.86 32.92 46.69 27.81 Greiner SST P 22.72 91.98 35.63 31.61 45.75 42.36 24.09 27.44 44.43 23.88 SST Tubes % Dose Difference Avg Greiner SST vs 56% 52% 80% 97% 61% 64% 69% 68% 55% 40% 64% BD SSTGreiner SST P vs 30% 29% 48% 57% 51% 38% 41% 40% 48% 20% 40% BD SSTLiHep Tubes % Dose DifferenceGreiner LiHep Gel -10% -3% 0% 2% 4% 6% -5% 16% 7% 3% 2% vs BD LiHep GelGreiner LiHep Gel 2% -3% 3% 1% 11% 1% 6% 14% 13% 5% 6%vs BD LiHep

[0112] It was determined that PVP K90 was the chemical responsible for the Greiner tube type bias. In order to overcome this tube type dose bias, the present disclosure optionally added PVP to the at least one releasing agent in the displacement reagent to saturate out this effect. A similar study was performed using the 2x ANS / 2x AE reagents but with increasing amounts of PVP added to the displacement reagent. Table 4 shows three samples in the absence of PVP, while Table 5 shows the samples with 0.01 mg / mL or 0.1 mg / mL PVP added to the displacement reagent. The dose values represented in Tables 4-5 are in ng / mL.TABLE 4: No PVPDonor 1 2 3BD LiHep 18.96 71.53 32.28BD LiHep Gel 19.93 74.75 31.18BD Red Top 14.54 65.68 27.80BD SST 16.84 72.35 31.94Greiner LiHep Gel 22.07 77.41 31.97Greiner SST 25.57 103.65 54.50Greiner SST P 23.55 92.24 48.31Greiner SST Tubes % Dose Difference Avg Greiner SST vs BD SST 52% 43% 71% 55% Greiner SST P vs BD SST 40% 27% 51% 40% Greiner LiHep Tubes % Dose DifferenceGreiner LiHep Gel vs BD LiHep Gel 11% 4% 3% 6% Greiner LiHep Gel vs BD LiHep 16% 8% -1% 8%BD Tube Types % Dose DifferenceBD LiHep vs BD Red Top 30% 9% 16% 18% BD SST vs BD Red Top 16% 10% 15% 14%% CM Across all tubes 19% 17% 28% 21%TABLE 5: Added PVP0.01 mg / mL PVP 0.1 mg / mL PVP Donor 1 2 3 1 2 3 BD LiHep 13.89 68.71 27.44 13.37 64.06 25.27 BD LiHep Gel 11.48 68.34 26.07 15.53 69.16 27.37 BD Red Top 10.53 63.96 24.31 14.24 63.59 23.49 BD SST 10.01 64.91 27.03 11.99 62.27 21.64 Greiner LiHep Gel 13.88 72.64 30.55 12.57 68.88 30.83 Greiner SST 12.75 71.64 27.98 12.54 70.03 27.81 Greiner SST P 12.4 69.57 27.10 11.9 63.42 29.63Greiner SST Tubes % Dose Difference Avg Avg Greiner SST vs BD SST 27% 10% 4% 14% 5% 12% 29% 15% Greiner SST P vs BD SST 24% 7% 0% 10% -1% 2% 37% 13% Greiner LiHep Tubes % Dose DifferenceGreiner LiHep Gel vs BD LiHep Gel 21% 6% 17% 15% -19% 0% 13% -2% Greiner LiHep Gel vs BD LiHep 0% 6% 11% 6% -6% 8% 22% 8%BD Tube Types % Dose DifferenceBD LiHep vs BD Red Top 32% 7% 13% 17% -6% 1% 8% 1% BD SST vs BD Red Top -5% 1% 11% 3% -16% -2% -8% -9%% CV Across all tubes 13% 5% 7% 8% 10% 5% 12% 9%

[0113] As can be seen in Tables 4 and 5, the addition of PVP to the displacement reagent decreased the average bias observed for Greiner SST tubes from 55% in the absence of PVPto 14% in the presence of 0.01 mg / mL PVP and 15% in the presence of 0.1 mg / mL PVP. Also, the average bias observed for Geiner SST P tubes decreased from 40% in the absence of PVP to 10% in the presence of 0.01 mg / mL PVP and 13% in the presence of 0.1 mg / mL PVP.

[0114] In addition, it was hypothesized that the PVP present in the Greinertubes may also interact with the FITC analog. Therefore, in order to overcome the tube type dose bias, the present disclosure prebound the vitamin D analog-containing conjugate and the PMPs prior to addition of sample or any other reagent to thereby provide a preformed second reagent that includes the vitamin D analog indirectly bound to the PMP. Then a similar study to that shown in Tables 4-5 was performed using the 2x ANS / 2x AE reagents with a preformed second reagent and in the absence or presence of increasing amounts of PVP added to the displacement reagent. Table 6 shows three samples analyzed with the preformed second reagent in the absence of PVP, while Table 7 shows the samples analyzed with the preformed second reagent and with 0.01 mg / mL or 0.1 mg / mL PVP added to the third / displacement reagent. The dose values represented in Tables 6-7 are in ng / mL.TABLE 6: Preformed Second Reagent and No PVPDonor 1 2 3BD LiHep 15.71 74.06 27.8BD LiHep Gel 17.24 77.48 26.19BD Red Top 17.98 74.91 26.89BD SST 17.38 73.58 26.65Greiner LiHep Gel 16.2 78.41 28.96Greiner SST 19.78 82.97 35.26Greiner SST P 19.04 79.63 30.85Greiner SST Tubes % Dose Difference AvgGreiner SST vs BD SST 14% 13% 32% 20% Greiner SST P vs BD SST 10% 8% 16% 11%Greiner LiHep Tubes % Dose DifferenceGreiner LiHep Gel vs BD LiHep Gel -6% 1% 11% 2% Greiner LiHep Gel vs BD LiHep 3% 6% 4% 4%BD Tube Types % Dose DifferenceBD LiHep vs BD Red Top -13% -1% 3% -3% BD SST vs BD Red Top -3% -2% -1% -2%% CV Across all tubes 8% 4% 11% 8%TABLE 7: Preformed Second Reagent and Added PVP0.01 mg / mL PVP 0.1 mg / mL PVP Donor 1 2 3 1 2 3BD LiHep 15.7 80.4 24.9 18.46 79.8 29.43 BD LiHep Gel 16.6 78.8 25.4 18.67 78.92 29.01 BD Red Top 15.6 73.2 24.4 18.48 74.12 28.19 BD SST 16.5 68.4 26.5 16.6 73.34 29.41 Greiner LiHep Gel 15.6 83.1 26.3 17.69 79.33 29.6 Greiner SST 15.8 79 26 17.28 76.33 31.63 Greiner SST P 15.6 74.3 28.4 16.48 76.85 31.62Greiner SST Tubes % Dose Difference Avg Avg Greiner SST vs BD SST -4% 15% -2% 3% 4% 4% 8% 5% Greiner SST P vs BD SST -5% 9% 7% 3% -1% 5% 8% 4% Greiner LiHep Tubes % Dose DifferenceGreiner LiHep Gel vs BD LiHep Gel -6% 5% 4% 1% -5% 1% 2% -1% Greiner LiHep Gel vs BD LiHep -1% 3% 6% 3% -4% -1% 1% -1%BD Tube Types % Dose DifferenceBD LiHep vs BD Red Top 1% 10% 2% 4% 0% 8% 4% 4% BD SST vs BD Red Top 6% -7% 9% 3% -10% -1% 4% -2%% CV Across all tubes 3% 7% 5% 5% 5% 3% 4% 4%

[0115] When the results of Tables 6-7 are compared to Tables 4-5, it is evident that preforming the second reagent decreased the average bias observed for Greiner SST tubes from 55% when two separate reagents were used to 20% when a preformed second reagent was utilized; in addition, this average bias decreased further to 3-5% in the presence of 0.01 -0.1 mg / mL PVP. Also, the average bias observed for Geiner SST P tubes decreased from 40% when two separate reagents were used to 11% when a preformed second reagent was used, and this bias decreased further to 3-4% in the presence of 0.01 - 0.1 mg / mL PVP.Example 2

[0116] The current Vitamin D assay over recovers samples containing high levels of 25(OH)vitamin D2 when the same sample contains low levels of 25(OH)vitamin D3. To minimize the 25(OH)vitamin D2over recovery, this Example found that a shorter incubation time of the pretreated sample with the anti-vitamin D antibody AE conjugate reduced 25(OH)vitamin D2doses while maintaining 25(OH)vitamin D3 doses, resulting in greater equimolarity between the two forms of vitamin D.

[0117] The purpose of this Example was to reduce the assay's over recovery of 25(OH)vitamin D2so that it aligns to the 25(OH)vitamin D3 doses when compared to LCMS (reference method). In these experiments, a panel of D2only samples was produced, and thena panel of D3 only samples spanning the same assay range was produced; both of these panels were assayed in the disclosed assay, and the obtained results were compared to LCMS values obtained with the panels. The data in FIGS. 3-5 one non-limiting set of exemplary results obtained with 25(OH)vitamin D2 and 25(OH)vitamin D3 serum panels tested with different incubation protocols and compared to LCMS values.

[0118] FIG. 3 contains the results of the control, which utilized the traditional six-minute incubation period for the pretreated sample with the anti-vitamin D antibody AE conjugate. The Slope Difference (D2 vs D3) for this control was 0.37 (i.e., almost a 40% variation). In contrast, FIGS. 4-5 contain the results obtained when the incubation time for the pretreated sample with the anti-vitamin D antibody AE conjugate was reduced to one minute; in addition, FIGS. 4-5 differ from one another based on the amount of pretreatment reagent utilized prior to addition of the anti-vitamin D antibody AE conjugate (2x ANS in FIG. 4 versus 2.5 ANS in FIG. 5). The Slope Difference (D2 vs D3) for these Figures was -0.01 for FIG. 4 (1 minute and the use of 2x ANS) and 0.03 for FIG. 5 (1 minute incubation and the use of 2.5x ANS); therefore, the slope difference with 1 minute incubation periods was less than 5%. As such, the difference in slope is reduced as the incubation time is shortened, indicating that the antibody is detecting the two different forms of vitamin D at the same rate.

[0119] Table 8 also discloses additional Slope Difference (D2 vs D3) values obtained at different incubation times. The slope difference trends closer to zero as the incubation time is shortened. A 2-minute incubation time has a slope difference of over 10%; however, an incubation time of about 1.5-minutes or less provides a less than 10% slope difference between D2 vs D3. Based on this data, an incubation time range of from about 0.5 minutes to about 1.5 minutes is indicated as providing a slope difference within ±10%.TABLE 8Incubation Period for PretreatedSlope Difference (D2 vs D3) Sample with Lite Reagent (antibody)8 minutes 0.436 minutes 0.37 -0.394 minutes 0.302 minutes 0.141 minute -0.01 -0.03NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0120] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0121] Illustrative embodiment 1. A kit for performing an immunoassay for Vitamin D, the kit comprising: a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and a second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent.

[0122] Illustrative embodiment 2. The kit of illustrative embodiment 1, further comprising a third reagent comprising at least one releasing agent.

[0123] Illustrative embodiment 3. The kit of illustrative embodiment 2, wherein the at least one releasing agent is selected from the group consisting of 8-anilino-1-naphthalene sulfonate (ANS), dithiothreitol (DTT), TRIS (2-carboxyethyl) phosphine (TCEP), sodium hydroxide (NaOH), and combinations thereof.

[0124] Illustrative embodiment 4. The kit of illustrative embodiment 2 or 3, wherein the at least one releasing agent comprises 8-anilino-1-naphthalene sulfonate (ANS).

[0125] Illustrative embodiment 5. The kit of any of illustrative embodiments 2-4, wherein the third reagent further comprises polyvinylpyrrolidone (PVP).

[0126] Illustrative embodiment 6. The kit of any of illustrative embodiments 2-5, wherein the third reagent further comprises at least one of ethylene glycol and / or methanol.

[0127] Illustrative embodiment 7. The kit of any of illustrative embodiments 1-6, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0128] Illustrative embodiment 8. The kit of illustrative embodiment 7, wherein the first label is an acridinium ester.

[0129] Illustrative embodiment 9. The kit of any of illustrative embodiments 1-8, wherein the second label is selected from the group consisting of fluorescein, biotin, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0130] Illustrative embodiment 10. The kit of illustrative embodiment 9, wherein the second label is fluorescein, and wherein the antibody or antigen-binding fragment thereof conjugated to the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0131] Illustrative embodiment 11. The kit of illustrative embodiment 10, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0132] Illustrative embodiment 12. The kit of any of illustrative embodiments 1-11, wherein the vitamin D analog is selected from the group consisting of 24R-Calcipotriol, Lythgoe diol 4-TBDMS ether, 25-Hydroxyvitamin D33-Hemisuccinate, Calcitriol-d6, Lythgoe diol, 25-OH vitamin D3-DPEG4-Biotin, 25-OH vitamin D₃-LC-Biotin, and vitamin D-C22 acid.

[0133] Illustrative embodiment 13. The kit of any of illustrative embodiments 1-12, wherein the vitamin D analog comprises a vitamin D-C22 analog.

[0134] Illustrative embodiment 14. The kit of any of illustrative embodiments 1-13, wherein the solid support comprises paramagnetic particles.

[0135] Illustrative embodiment 15. The kit of any of illustrative embodiments 1-14, wherein the kit detects 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

[0136] Illustrative embodiment 16. A kit for performing an immunoassay for Vitamin D, the kit comprising: a first reagent comprising an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragmentthereof labeled with acridinium ester; a second reagent comprising a conjugate of a vitamin D analog and fluorescein and paramagnetic particles coated with anti-fluorescein monoclonal antibody or an antigen-binding fragment thereof, wherein the fluorescein is bound to the anti-fluorescein antibody / binding fragment thereof, and wherein the monoclonal antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent

[0137] Illustrative embodiment 17. The kit of illustrative embodiment 16, further comprising a third reagent comprising at least one releasing agent.

[0138] Illustrative embodiment 18. The kit of illustrative embodiment 17, wherein the at least one releasing agent is selected from the group consisting of 8-anilino-1-naphthalene sulfonate (ANS), dithiothreitol (DTT), TRIS (2-carboxyethyl) phosphine (TCEP), sodium hydroxide (NaOH), and combinations thereof.

[0139] Illustrative embodiment 19. The kit of illustrative embodiment 17 or 18, wherein the at least one releasing agent comprises 8-anilino-1-naphthalene sulfonate (ANS).

[0140] Illustrative embodiment 20. The kit of any of illustrative embodiments 17-19, wherein the third reagent further comprises polyvinylpyrrolidone (PVP).

[0141] Illustrative embodiment 21. The kit of any of illustrative embodiments 17-20, wherein the third reagent further comprises at least one of ethylene glycol and / or methanol.

[0142] Illustrative embodiment 22. The kit of any of illustrative embodiments 17-21, wherein the vitamin D analog is selected from the group consisting of 24R-Calcipotriol, Lythgoe diol 4-TBDMS ether, 25-Hydroxyvitamin D33-Hemisuccinate, Calcitriol-d6, Lythgoe diol, 25-OH vitamin D3-DPEG4-Biotin, 25-OH vitamin D₃-LC-Biotin, and vitamin D-C22 acid.

[0143] Illustrative embodiment 23. The kit of any of illustrative embodiments 17-22, wherein the vitamin D analog comprises a vitamin D-C22 analog.

[0144] Illustrative embodiment 24. The kit of any of illustrative embodiments 17-23, wherein the kit detects 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

[0145] Illustrative embodiment 25. A system for performing an immunoassay for Vitamin D, the system comprising: the kit of any of illustrative embodiments 1-24; and a sample collection tube.

[0146] Illustrative embodiment 26. The system of illustrative embodiment 25, wherein the sample collection tube is a serum separator tube.

[0147] Illustrative embodiment 27. The system of illustrative embodiment 25 or 26, wherein the sample collection tube comprises PVP.

[0148] Illustrative embodiment 28. The system of any of illustrative embodiments 25-27, further comprising at least one additional reagent for initiating detection of the chemiluminescence triggered by the acridinium ester.

[0149] Illustrative embodiment 29. The system of illustrative embodiment 28, wherein the at least one additional reagent comprises an acid and a base.

[0150] Illustrative embodiment 30. A method of determining a concentration of vitamin D in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigenbinding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent; (2) incubating the mixture under conditions whereby (b) binds to vitamin D present in the biological sample; (3) detecting a signal generated by the first label indirectly bound to the solid support; and (4) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label indirectly bound to the solid support.

[0151] Illustrative embodiment 31. A method of determining a concentration of vitamin D in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigenbinding fragment thereof coated on the solid support, and wherein the antibody or antigenbinding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent; (2) incubating the mixture under conditions whereby (b) binds to vitamin D present in the biological sample; (3) detecting a signal generated by the first label on the solid support; and (4) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0152] Illustrative embodiment 32. A method of detecting vitamin D deficiency in a subject, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample derived from the subject; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigenbinding fragment thereof coated on the solid support, and wherein the antibody or antigenbinding fragment thereof of the first reagent specifically binds to the vitamin D analog of thesecond reagent; (2) incubating the mixture under conditions whereby (b) binds to vitamin D present in the biological sample; (3) detecting a signal generated by the first label on the solid support; and (4) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0153] Illustrative embodiment 33. The method of any of illustrative embodiments 30-32, wherein a decrease between the level of vitamin D in the biological sample relative to the level in a normal control or a threshold level of 30 ng / mL is indicative of a vitamin D deficiency in said subject.

[0154] Illustrative embodiment 34. A method for treating a subject suspected of having a vitamin D deficiency, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture: (a) a biological sample derived from the subject; (b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and (c) a preformed second reagent comprising a conjugate of a second label and a vitamin D analog and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent; (2) incubating the mixture under conditions whereby (b) binds to vitamin D present in the biological sample; (3) detecting a signal generated by the first label on the solid support; (4) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support; and (5) if a decrease between the level of vitamin D in the biological sample relative to the level in a normal control or a threshold level of 30 ng / mL is determined, administering to said subject a treatment for vitamin D deficiency.

[0155] Illustrative embodiment 35. A method for monitoring progression of vitamin D deficiency in a subject in need thereof, said method comprising the steps of: (i) performing steps (1)-(4) of any of illustrative embodiments 30-34 using a first biological sample collected at a first time point; (ii) performing steps (1)-(4) of any of illustrative embodiments 30-34 using a second biological sample collected at a second time point after the first time point; and wherein a decrease between the level of vitamin D present in the first biological sample andthe level of vitamin D present in the second biological sample is indicative of the progression of a vitamin D deficiency in said subject, wherein little or no change between the levels of vitamin D in the first and second biological samples is indicative of stabilization of a vitamin D deficiency in the subject, and wherein an increase between the level of vitamin D in the first biological sample and the level of vitamin D in the second biological sample is indicative of regression of a vitamin D deficiency in the subject.

[0156] Illustrative embodiment 36. A method for monitoring treatment of vitamin D deficiency in a subject in need thereof, said method comprising the steps of: (i) performing steps (1)-(4) of any of illustrative embodiments 30-34 using a first biological sample collected at a first time point; (ii) performing steps (1)-(4) ofany of illustrative embodiments 30-34 using a second biological sample collected at a second time point after the first time point and following administration to the subject of a treatment for vitamin D deficiency; and wherein an increase in or stabilization of the level of vitamin D in the second biological sample relative to the level of vitamin D in the first biological sample is indicative of efficacy of the treatment of the vitamin D deficiency in said subject, and wherein a decrease in the level of vitamin D in the second biological sample relative to the level of vitamin D in the first biological sample is indicative of inefficacy of the treatment of the vitamin D deficiency in said subject.

[0157] Illustrative embodiment 37. The method of any of illustrative embodiments 30-36, wherein the biological sample is at least one of blood, serum, or plasma.

[0158] Illustrative embodiment 38. The method of any of illustrative embodiments 30-37, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

[0159] Illustrative embodiment 39. The method of illustrative embodiment 38, wherein the sample collection tube is a serum separator tube.

[0160] Illustrative embodiment 40. The method of any of illustrative embodiments 30-39, further comprising performing a wash step prior to step (3).

[0161] Illustrative embodiment 41. The method ofany of illustrative embodiments 30-40, wherein the first label is acridinium ester, and wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0162] Illustrative embodiment 42. The method of any of illustrative embodiments 30-41, wherein the second label of (c) is fluorescein.

[0163] Illustrative embodiment 43. The method of any of illustrative embodiments 30-42, wherein the antibody or antigen-binding fragment thereof conjugated to the solid support of (c) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0164] Illustrative embodiment 44. The method of any of illustrative embodiments 30-43, wherein the vitamin D analog of (c) is selected from the group consisting of 24R-Calcipotriol, Lythgoe diol 4-TBDMS ether, 25-Hydroxyvitamin D33-Hemisuccinate, Calcitriol-d6, Lythgoe diol, 25-OH vitamin D3-DPEG4-Biotin, 25-OH vitamin D3-LC-Biotin, and vitamin D-C22 acid.

[0165] Illustrative embodiment 45. The method of any of illustrative embodiments 30-44, wherein the vitamin D analog of (c) comprises a vitamin D-C22 analog.

[0166] Illustrative embodiment 46. The method of any of illustrative embodiments 30-45, wherein the solid support of (c) comprises paramagnetic particles.

[0167] Illustrative embodiment 47. The method of any of illustrative embodiments 30-46, wherein step (1) further includes adding (d) a third reagent that comprises at least one releasing agent.

[0168] Illustrative embodiment 48. The method of illustrative embodiment 47, wherein the at least one releasing agent of (d) is selected from the group consisting of 8-anilino-1-naphthalene sulfonate (ANS), dithiothreitol (DTT), TRIS (2-carboxyethyl) phosphine (TCEP), sodium hydroxide (NaOH), and combinations thereof.

[0169] Illustrative embodiment 49. The method of any of illustrative embodiments 47-48, wherein the at least one releasing agent of (d) comprises 8-anilino-1-naphthalene sulfonate (ANS).

[0170] Illustrative embodiment 50. The method of any of illustrative embodiments 47-49, wherein the third reagent of (d) further comprises polyvinylpyrrolidone (PVP).

[0171] Illustrative embodiment 51. The method of any of illustrative embodiments 47-50, wherein the third reagent of (d) further comprises at least one of ethylene glycol and / or methanol.

[0172] Illustrative embodiment 52. The method of any of illustrative embodiments 47-51, wherein in step (1), (a) and (d) are combined prior to addition of (b) and (c).

[0173] Illustrative embodiment 53. The method of any of illustrative embodiments 30-52, wherein the method detects a concentration of 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

[0174] Illustrative embodiment 54. A method of determining a concentration of vitamin D in a biological sample, the method comprising the steps of: (1) combining a biological sample with a reagent comprising at least one releasing agent to form a mixture; (2) adding a reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof to form an antibody-containing mixture; (3) incubating the antibody-containing mixture for a period in a range of from about 30 seconds to about 90 seconds and under conditions whereby the anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof binds to vitamin D present in the biological sample; (4) adding to the antibody-containing mixture a reagent comprising a conjugate of a second label and a vitamin D analog, and a solid support coated with an antibody or antigenbinding fragment thereof that specifically binds to the second label, and wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent; (5) detecting a signal generated by the first label on the solid support; and (6) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

[0175] Illustrative embodiment 55. The method of Illustrative embodiment 54, wherein the method detects 25-hydroxyvitamin D2and 25-hydroxyvitamin D3at substantially the same rate.

[0176] Illustrative embodiment 56. The method of Illustrative embodiment 54 or 55, wherein the reagent of step (1) further comprises polyvinylpyrrolidone (PVP).

[0177] Illustrative embodiment 57. The method of any of Illustrative embodiments 54-56, wherein the biological sample is at least one of blood, serum, or plasma.

[0178] Illustrative embodiment 58. The method of any of Illustrative embodiments 54-57, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

[0179] Illustrative embodiment 59. The method of Illustrative embodiment 58, wherein the sample collection tube is a serum separator tube.

[0180] Illustrative embodiment 60. The method of any of Illustrative embodiments 54-59, further comprising performing a wash step prior to step (5).

[0181] Illustrative embodiment 61. The method of any of Illustrative embodiments 54-60, wherein the first label is acridinium ester, and wherein step (5) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0182] Illustrative embodiment 62. The method of any of Illustrative embodiments 54-61, wherein the second label of (a) is fluorescein, and the antibody or antigen-binding fragment thereof conjugated to the solid support of (b) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0183] Illustrative embodiment 63. The method of any of Illustrative embodiments 54-62, wherein the vitamin D analog of (a) comprises a vitamin D-C22 analog.

[0184] Illustrative embodiment 64. The method of any of Illustrative embodiments 54-63, wherein the solid support of (b) comprises paramagnetic particles.

[0185] Illustrative embodiment 65. The method of any of Illustrative embodiments 54-64, wherein the at least one releasing agent utilized in step (1) comprises 8-anilino-1-naphthalene sulfonate (ANS).

[0186] Illustrative embodiment 66. The method of any of Illustrative embodiments 54-65, wherein the reagent comprising the at least one releasing agent further comprises at least one of ethylene glycol and / or methanol.

[0187] Thus, in accordance with the present disclosure, there have been provided methods, devices, systems, and / or apparatus which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

CLAIMS1. A kit for performing an immunoassay for Vitamin D, the kit comprising:a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and a second reagent comprising:a conjugate of a second label and a vitamin D analog; anda solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label; andwherein the second label of the conjugate is bound to the antibody or antigenbinding fragment thereof coated on the solid support; and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent.

2. The kit of claim 1, further comprising a third reagent comprising at least one releasing agent.

3. The kit of claim 2, wherein the at least one releasing agent comprises 8-anilino-1-naphthalene sulfonate (ANS).

4. The kit of claim 2, wherein the third reagent further comprises polyvinylpyrrolidone (PVP).

5. The kit of claim 2, wherein the third reagent further comprises at least one of ethylene glycol and / or methanol.

6. The kit of claim 1, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

7. The kit of claim 6, wherein the first label is an acridinium ester.

8. The kit of claim 1, wherein the second label is selected from the group consisting of fluorescein, biotin, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

9. The kit of claim 8, wherein the second label is a fluorescein, and wherein the antibody or antigen-binding fragment thereof conjugated to the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

10. The kit of claim 9, wherein the fluorescein is fluorescein isothiocyanate (FITC).

11. The kit of claim 1, wherein the vitamin D analog of the second reagent comprises a vitamin D-C22 analog.

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

13. The kit of claim 1, wherein the kit detects 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

14. A system for performing an immunoassay for Vitamin D, the system comprising: the kit of any of claims 1-13; anda sample collection tube.

15. The system of claim 14, wherein the sample collection tube is a serum separator tube.

16. The system of claim 14, wherein the sample collection tube comprises PVP.

17. The system of claim 14, further comprising at least one additional reagent for initiating detection of the first label.

18. A method of determining a concentration of vitamin D in a biological sample, the method comprising the steps of:(1) combining, either simultaneously or wholly or partially sequentially, (a)-(c) to form a mixture:(a) a biological sample;(b) a first reagent comprising a first label conjugated to an anti-25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof; and(c) a second reagent comprising a conjugate of a second label and a vitamin D analog, and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, and wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent;(2) incubating the mixture under conditions whereby (b) binds to vitamin D present in the biological sample;(3) detecting a signal generated by the first label on the solid support; and (4) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

19. The method of claim 18, wherein the biological sample is at least one of blood, serum, or plasma.

20. The method of claim 18, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

21. The method of claim 20, wherein the sample collection tube is a serum separator tube.

22. The method of claim 18, further comprising performing a wash step prior to step (3).

23. The method of claim 18, wherein the first label is acridinium ester, and wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

24. The method of claim 18, wherein at least one of:the second label of (c) is fluorescein, and the antibody or antigen-binding fragment thereof conjugated to the solid support of (c) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof;the vitamin D analog of (c) comprises a vitamin D-C22 analog; and / orthe solid support of (c) comprises paramagnetic particles.

25. The method of claim 18, wherein step (1) further includes adding (d) a third reagent comprising at least one releasing agent and polyvinylpyrrolidone (PVP).

26. The method of claim 25, wherein the at least one releasing agent comprises 8-anilino-1-naphthalene sulfonate (ANS).

27. The method of claim 25, wherein the third reagent of (d) further comprises at least one of ethylene glycol and / or methanol.

28. The method of claim 25, wherein (a) and (d) are combined prior to addition of (b)-(c).

29. The method of claim 18, wherein the method detects a concentration of 25-hydroxyvitamin D2 and / or 25-hydroxyvitamin D3.

30. A method of determining a concentration of vitamin D in a biological sample, the method comprising the steps of:(1) combining a biological sample with a reagent comprising at least one releasing agent to form a mixture;(2) adding a reagent comprising a first label conjugated to an anti-25- hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof to form an antibody-containing mixture;(3) incubating the antibody-containing mixture for a period in a range of from about 30 seconds to about 90 seconds and under conditions whereby the anti- 25-hydroxyvitamin D monoclonal antibody or antigen-binding fragment thereof binds to vitamin D present in the biological sample;(4) adding to the antibody-containing mixture a reagent comprising a conjugate of a second label and a vitamin D analog, and a solid support coated with an antibody or antigen-binding fragment thereof that specifically binds to the second label, and wherein the second label of the conjugate is bound to the antibody or antigen-binding fragment thereof coated on the solid support, and wherein the antibody or antigen-binding fragment thereof of the first reagent specifically binds to the vitamin D analog of the second reagent;(5) detecting a signal generated by the first label on the solid support; and (6) determining a concentration of vitamin D present in the biological sample, wherein the concentration is inversely proportional to the amount of signal generated by the first label on the solid support.

31. The method of claim 30, wherein the method detects 25-hydroxyvitamin D2and 25-hydroxyvitamin D3at substantially the same rate.

32. The method of claim 30, wherein the reagent of step (1) further comprises polyvinylpyrrolidone ( PVP).

33. The method of claim 30, wherein the biological sample is at least one of blood, serum, or plasma.

34. The method of claim 30, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube, and optionally wherein the sample collection tube is a serum separator tube.

35. The method of claim 30, further comprising performing a wash step prior to step (5).

36. The method of claim 30, wherein at least one of:the first label is acridinium ester, and wherein step (5) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs);the second label is fluorescein, and the antibody or antigen-binding fragment thereof conjugated to the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof;the vitamin D analog comprises a vitamin D-C22 analog;the solid support comprises paramagnetic particles;the at least one releasing agent utilized in step (1) comprises 8-anilino-1-naphthalene sulfonate (ANS); and / orthe reagent comprising the at least one releasing agent further comprises at least one of ethylene glycol and / or methanol.

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