Compositions, kits, and systems for performing immunoassays with preformed solid reagents and methods of producing and using same

Abstract

Compositions, reagents, kits, systems, and methods for performing immunoassays for a target analyte are disclosed. The compositions, reagents, kits, and systems utilize a preformed solid phase reagent that includes a streptavidin-coated solid support conjugated to a biotinylated antibody or antigen-binding fragment thereof that specifically binds to a label of another reagent. The biotinylated antibody or antigen-binding fragment is bound to the solid support during the manufacturing process so that any interference in the immunoassay by biotin present in a biological sample is mitigated.

Description

Atty. 2024P19111USTITLE COMPOSITIONS, KITS, AND SYSTEMS FOR PERFORMING IMMUNOASSAYS WITH PREFORMED SOLID REAGENTS AND METHODS OF PRODUCING AND USING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 755,375, filed February 7, 2025. 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.BACKGROUND

[0003] Diagnostic assay reagents often include conjugates of antibodies or other small drug molecules with haptens, such as biotin and fluorescein. The tight binding of these haptens to large protein molecules (e.g., avidin / streptavidin for biotin and anti-FITC for fluorescein) that are coated on a solid support or surface provides a convenient way to immobilize the haptenantibody or hapten-drug conjugate on the solid support / surface.

[0004] Biotin is well known in the art for its use as a food supplement; for example, biotin is utilized to promote healthy hair and nail growth and to treat various disease conditions. Given this use, significant biotin levels can be found in biological samples, such as (but not limited to) blood. Since biotin is used in many diagnostic assays (for example, to coat solid supports), high levels of biotin in test samples can interfere with assay signals in any assays where biotinylated assay components are employed. This is particularly true for assays where biotinylated assay components are expected to bind streptavidin-coated solid supports.

[0005] Therefore, there is a need in the art for new and improved compositions, reagents, kits, and systems for use in immunoassays that overcome the disadvantages and defects of the prior art. It is to such compositions, reagents, kits, and systems, as well as methods of producing and using same, that the present disclosure is directed.Atty. 2024P19111USBRIEF DESCRIPTIONS OF THE DRAWINGS

[0006] FIG. 1 schematically depicts Hepatitis B surface antigen (HBsAg) immunoassay format of the prior art.

[0007] FIG. 2 schematically illustrates one non-limiting embodiment of an immunoassay architecture constructed in accordance with the present disclosure.

[0008] FIG. 3 schematically illustrates an immunoassay performed using the reagents of FIG.2.

[0009] FIG. 4 schematically illustrates one non-limiting embodiment of an HBsAg immunoassay format constructed in accordance with the present disclosure.

[0010] FIG. 5 schematically illustrates one non-limiting embodiment of a competitive immunoassay architecture constructed in accordance with the present disclosure.

[0011] FIG. 6 schematically illustrates another non-limiting embodiment of a competitive immunoassay architecture constructed in accordance with the present disclosure.DETAILED DESCRIPTION

[0012] 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.

[0013] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0014] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill intheart. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include the singular. The foregoing techniques and proceduresAtty. 2024P19111USare 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.

[0015] 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.

[0016] 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.

[0017] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0018] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / orthe specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example,Atty. 2024P19111USreference 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."

[0019] 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).

[0020] 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.

[0021] 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.Atty. 2024P19111USFor example, a condition " A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0022] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with 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.

[0023] 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 over time, 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, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0024] 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, unlessAtty. 2024P19111USotherwise 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 itAtty. 2024P19111USis 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".

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 asAtty. 2024P19111USpeptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than 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 well known in the art. Such modifications are well described in basic texts, monographs, and research literature. Modifications may occur anywhere in a polypeptide, including 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, gamma-carboxylation, 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).

[0034] The term "biomolecules" includes proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof.Atty. 2024P19111US

[0035] 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 fragment thereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.

[0036] 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.

[0037] 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) oran 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(ab')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 orconjugates 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).

[0038] 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 anAtty. 2024P19111USantigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, 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 are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[0039] 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.

[0040] 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.

[0041] 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 cross-species 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).

[0042] 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 includeAtty. 2024P19111USchemically 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.

[0043] 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 certain embodiments, 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.

[0044] 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).

[0045] 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 thanAtty. 2024P19111US95% 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 nonreducing 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 having different antigenic specificities. An isolated antibody may, however, have some cross-reactivity to other, related antigens.

[0046] 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 either the 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%.

[0047] 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 comprisingthe 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)).Atty. 2024P19111US

[0048] 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.

[0049] 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.

[0050] An "analyte" is a molecule that is capable of being recognized by an analyte-specific 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 analytespecific binding partner (i.e., antibody).

[0051] 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, nonAtty. 2024P19111UScovalent 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.

[0052] 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.

[0053] Software may include one or more computer readable instructions that when executed by one or more processing components cause the processing component to perform 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.

[0054] 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.

[0055] 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, aAtty. 2024P19111USnetwork interface, a human, a user terminal, a virtual computer, combinations thereof, and the like, for example.

[0056] 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.

[0057] 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.

[0058] 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 or any portion thereof (i.e., plasma orserum), 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.

[0059] 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 immunoassays, as well as methods of producing and using same. The immunoassays utilize at least three reagents, including a preformed reagent that comprises a streptavidin-coated solid support and a biotinylated antibody or antigen-binding fragment thereof prebound thereto that specifically binds to a label attached to another reagent utilized in the immunoassay. The biotinylated antibody / fragment thereof is prebound to the streptavidin-coated solid support during the manufacturing process and prior to contact with biological sample. The immunoassay may have a competitive or non-competitive format. By utilizing the preformed reagent in which the biotinylated antibody / fragment thereof is preboundAtty. 2024P19111USto the streptavidin-coated solid support, any potential interference from biotin present in a biological sample being assayed is mitigated.

[0060] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing an immunoassay for a target analyte that utilizes at least three reagents. The first reagent includes a conjugate of a first label and a first antibody or antigen-binding fragment thereof that specifically binds to the target analyte, while the second reagent includes a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte. The third reagent is the prebound reagent described herein above and includes a streptavidin-coated solid support and a third antibody or antigenbindingfragment thereof that specifically binds to the second label; the third antibody or antigenbinding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigen-binding fragment thereof to the solid support. The first and second antibodies or antigen-binding fragments thereof bind to substantially nonoverlapping epitopes of the target analyte so that the first and second reagents bind to the same target analyte molecule to form a sandwich composition. Also, the third antibody or antigenbinding fragment thereof of the third reagent binds to the second label ofthe second reagent to attach the sandwich composition to the solid support. The amount of target analyte present in a biological sample is directly proportional to the amount of signal generated by the first label when it is indirectly bound to the solid support that is detected by the system.

[0061] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing a competitive immunoassay for a target analyte, wherein the kit includes three reagents. The first reagent includes a conjugate of a first label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte, while the second reagent includes a conjugate of a second label and a target analyte or analog thereof to which the antibody or antigen-binding fragment thereof can specifically bind. The third, prebound reagent includes a streptavidin-coated solid support and an antibody or antigen-binding fragment thereof that specifically binds to the second label, and the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin is bound to the streptavidin on the solid support such that the antibody or antigen-binding fragment thereof is attached to the solidAtty. 2024P19111USsupport. An inverse relationship exists between the amount of target analyte present in a 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] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing a competitive immunoassay for a target analyte, wherein the kit includes three reagents, a first reagent comprising a conjugate of a first label and a target analyte or analog thereof; a second reagent comprising a conjugate of a second label and an antibody or antigenbinding fragment thereof that specifically binds to the target analyte or analog thereof; and a third reagent comprising a streptavidin-coated solid support and an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the antibody or antigenbinding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support. An inverse relationship exists between the amount of target analyte 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.

[0063] Certain non-limiting embodiments of the present disclosure are directed to systems for performing competitive or non-competitive immunoassays for at least one target analyte, 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).

[0064] 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.

[0065] 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)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a conjugate of aAtty. 2024P19111USfirst label and a first antibody or antigen-binding fragment thereof that specifically binds to the target analyte; (c) a second reagent comprising a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte; and (d) a third reagent comprising a streptavidin-coated solid support having a third antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the third antibody or antigen-binding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigenbinding fragment thereof to the solid support, and wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte. In step (2), the mixture is incubated under conditions whereby (b) and (c) bind to target analyte present in the biological sample to form a sandwich composition, and the third antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d). In step (3), a signal generated by the first label of (b) on the solid support of (d) is detected. In step (4), a concentration of the target analyte present in the biological sample is determined, wherein the concentration is directly proportional to the amount of signal generated by the first label on the solid support.

[0066] Certain non-limiting embodiments of the present disclosure are directed to a method of determining a concentration of a target analyte in a biological sample utilizing a competitive immunoassay format. In the method, step (1) includes combining, either simultaneously or wholly or partially sequentially, the following components (a)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a conjugate of a first label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte; (c) a second reagent comprising a conjugate of a second label and a target analyte or analog thereof to which the antibody or antigen-binding fragment of (b) can specifically bind; and (d) a third reagent comprising a streptavidin-coated solid support having an antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support. In step (2), the mixture is incubated under conditions whereby, inAtty. 2024P19111USthe absence of target analyte present in the biological sample, (b) and (c) bind to form a sandwich composition, and the antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d); alternatively, when target analyte is present in the biological sample, (b) binds to the target analyte, and the sandwich composition does not form. In step (3), a signal generated by the first label of (b) on the solid support of (d) is detected. In step (4), a concentration of the target analyte 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.

[0067] Certain non-limiting embodiments of the present disclosure are directed to a method of determining a concentration of a target analyte in a biological sample utilizing a competitive immunoassay format. In the method, step (1) includes combining, either simultaneously or wholly or partially sequentially, the following components (a)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a conjugate of a first label and a target analyte or analog thereof; (c) a second reagent comprising a conjugate of a second label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte or analog thereof; and (d) a third reagent comprising a streptavidin-coated solid support having an antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support. In step (2), the mixture is incubated under conditions whereby, in the absence of target analyte present in the biological sample, (b) and (c) bind to form a sandwich composition, and the antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d); alternatively, in the presence of target analyte in the biological sample, (c) binds to the target analyte, and the sandwich composition does not form. In step (3), a signal generated by the first label of (b) on the solid support of (d) is detected. In step (4) a concentration of the target analyte 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.Atty. 2024P19111US

[0068] 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.

[0069] 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 prior to 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.

[0070] The components (a)-(d) (as well as any additional components, 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 (c) and (d) are combined priorto addition of the biological sample (a).

[0071] The kits, systems, and methods of the present disclosure may include one or more additional reagents containing one or more anti-analyte-specific antibodies or antigen-binding fragments thereof for providing more accurate concentration measurements. For example (but not by way of limitation), the use of the additional antibody-containing reagents may provide greater sensitivity across a wider concentration range. Alternatively, the use of additional antibody-containing reagents may allow for detection of multiple transcripts, variants, derivatives, analogs, and / or mutants within a single immunoassay.

[0072] Non-limiting examples of such additional reagents that may be utilized in accordance with the kits, systems, and methods for performing a non-competitive immunoassay of the present disclosure include: a fourth reagent that comprises a conjugate of the second label and a fourth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the first and fourth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the first and fourth reagents bind to the same target analyte molecule to form a sandwich composition, and the thirdAtty. 2024P19111USantibody or antigen-binding fragment thereof of the third reagent binds to the second label of the fourth reagent to attach the sandwich composition to the solid support; and / or a fifth reagent that comprises a conjugate of the first label and a fifth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the second and fifth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the second and fifth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support. Similar reagents can also be utilized in accordance with the kits, systems, and methods for performing a competitive immunoassay of the present disclosure.

[0073] The kits, systems, and methods of the present disclosure may be utilized to detect any target analyte in a biological sample that is capable for detection by a competitive or noncompetitive immunoassay, and for which at least one antibody is known for a competitive immunoassay or for which at least two antibodies are known that are capable of binding to substantially non-overlapping epitopes to form a sandwich complex, for a non-competitive immunoassay format. Non-limiting examples of target analytes that are capable of detection in this manner include Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM); Hepatitis A virus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; N-terminal peptide (PIIINP); and the like.

[0074] Antibodies for detection of the analytes listed above 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 such monoclonal antibodies or antigen-binding fragments thereof include Antibodies-Online Inc. (Limerick, PA); Creative Biolabs, Inc. (Shirley, NY); Creative Diagnostics (Shirley, NY); Immunodiagnostik AG (Bensheim, Germany); MyBioSource, Inc. (San Diego, CA); US Biological Life Sciences (Salem, MA); and many others. However, this list is not inclusive, and there are manyAtty. 2024P19111USadditional commercial sources of antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of a target analyte and one or more antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the analytes or antibodies or the characteristics thereof is deemed necessary.

[0075] In addition, as stated throughout the application, the antibodies utilized herein may be full length antibodies or portions thereof, such as antigen-binding fragments thereof. The use of antigen-binding fragments, such as (but not limited to) a F(ab')2 fragment, may mitigate or even eliminate interference from other molecules, such as (but not limited to) rheumatoid factors.

[0076] Any of the antibodies or antigen-binding fragments described or otherwise contemplated herein (as well as any of the target analytes or analogs thereof utilized in the competitive immunoassay format) may be labeled or otherwise conjugated to various chemical or biomolecule moieties for use in the diagnostic applications disclosed herein. The moieties may be detectable labels, for example (but not by way of limitation), chemiluminescent labels (e.g., acridinium esters and sulfonamides, luminol and isoluminol), phosphorescent labels, fluorescent labels (e.g., FITC), electrochemiluminescent labels (e.g., ruthenium (II) chelates), cloned enzyme donors, photosensitizer particles or chemiluminescer particles for luminescent oxygen channeling immunoassays (LOCI), lanthanide chelates for time-resolved fluorescence immunoassays (TR-FIA), radiolabels, biotin, digoxigenin, enzymes and the like, for example, radionuclides, such as, but not limited to, tritium, carbon-14, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111, and fissionable nuclides such as boron-10 or an actinide. In some embodiments, enzymes may be conjugated to the described antibodies or antigen-binding proteins (or target analytes / analogs thereof utilized in the competitive immunoassay format) 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 beAtty. 2024P19111USsuitable for use as a conjugate for the antibodies and antigen-binding fragments (or target analytes / analogs thereof utilized in the competitive immunoassay format) described herein. In addition, compounds such as acridinium esters may also be conjugated to the provided antibodies and antigen-binding fragments (or target analytes / analogs thereof utilized in the competitive immunoassay format) to allow for detection in an immunoassay.

[0077] In certain particular (but non-limiting) embodiments, the first label utilized in the first reagents of the kits, systems, and methods described or otherwise contemplated herein is a detectable label. For example (but not by way of limitation), the first label can be a chemiluminescent compound (e.g., an acridinium ester compound), a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and / or an enzyme. These exemplary labels can usually only be detected when excited by methods that include, but are not limited to, addition of different chemicals, stimulation by light, or exposure to substrate or other compounds. When using an acridinium ester compound, chemiluminescence is triggered by peroxide and acid / base, resulting in a flash that can be read by appropriate instrumentation. In addition, an optional wash step may be used before initiating detectability of the detectable label.

[0078] 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).

[0079] The second label utilized in the second reagents of the kits, systems, and methods described or otherwise contemplated herein may be any molecule for which an antibody or other specific binding partner is available that can be conjugated to the solid support, so long as the second label and the antibody / binding partner do not otherwise interfere with the assay being performed. Non-limiting examples of second labels and antibody / specific binding partner combinations that can be utilized in the kits, systems, and methods in accordance with the present disclosure include fluorescein (e.g., fluorescein isothiocyanate (FITC)) for binding to antifluorescein 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 partnerAtty. 2024P19111UScombinations; 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.

[0080] Any solid supports known in the art and capable of functioning in accordance with the kits, systems, and methods described herein may be utilized in accordance with the present disclosure. Non-limiting examples of solid phase supports that may be utilized include magnetic particles, such as (but not limited to) magnetic latex particles; paramagnetic particles (PMPs); cross-linked dextran available under the trademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N. J.); agarose; polystyrene beads; polyvinyl chloride; polystyrene; cross-linked polyacrylamide; nitrocellulose- or nylon-based webs such as sheets, strips, or paddles; or tubes, plates, or wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. When using paramagnetic particles, some source of a 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.

[0081] 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 non-limiting) 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.

[0082] Also, the compositions / reagents present in the kits or systems may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. In addition, the kit or system may include a device (such as, but not limited to, a microfluidics device or cartridge for loading onboard of a clinical instrument) in which one or more of the compositions / reagents are disposed.Atty. 2024P19111US

[0083] 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.

[0084] In addition to mitigating interference from biotin and rheumatoid factors, as discussed herein above, the compositions, reagents, kits, systems, and methods of the present disclosure may further mitigate interference from one or more other molecules, such as (but not limited to) hemoglobin, intralipids, total protein, bilirubin, and the like.EXAMPLES

[0085] 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.

[0086] FIG. 1 illustrates a currently available ADVIA CENTAUR® HBsAgll assay 10 (Siemens Healthineers USA, Malvern, PA), a magnetic particle chemiluminometric immunoassay for the qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum and plasma that can be utilized to diagnose and monitor individuals with acute or chronic hepatitis B infection or screen for hepatitis B infection in, for example, pregnant women. The assay 10 is a sandwich immunoassay using direct, chemiluminometric technology. The reagents include an ancillary pack reagent 20 that comprises multiple biotinylated anti-HBs mouse monoclonal captureAtty. 2024P19111USantibodies (indicated by reference numerals 22, 24, and 26, for purposes of example only) and an acridinium-ester-labeled anti-HBs mouse monoclonal antibody 28. Incubation with a biological sample allows HBsAg present in the sample (such as, but not limited to, AD / AY subtypes 30 and mutant HBsAg 32) to complex with two or more of the various antibodies 22, 24, 26, and 28 to form one or more complexes (indicated by reference numerals 40 and 42, for purposes of example only). A primary reagent pack 50 includes a solid reagent / streptavidin-coated magnetic latex particle 52 and may further include a light reagent / second acridinium-ester-labeled anti-HBs mouse monoclonal antibody 54 (to increase the signal generated). The particles 52 capture the sandwich complexes 40 and / or 42 to form one or more solid support-containing complexes 60, and a signal is generated by the acridinium esters of the conjugates 28 and 54 indirectly bound to the particles 52. A direct relationship exists between the amount of HBsAg activity present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0087] However, if biotin is present in a patient sample, the biotin will interfere with the binding of any of the biotinylated antibodies 22, 24, 26 to the streptavidin-coated microparticles 52. Because of the competitive inhibition of biotin to the biotin binding site of the microparticles 52, the patient results will be erroneous.

[0088] Therefore, to mitigate this interference by any biotin present in the patient sample, a new assay architecture 100 has been developed that can be utilized with any target analyte; one non-limiting embodiment of this new assay architecture 100 is shown in FIG. 2. The new assay architecture 100 includes a first reagent 102, a second reagent 104, and a third reagent 106. The first reagent 102 is a conjugate of a first antibody (or fragment thereof) 110 that specifically binds to the target analyte, conjugated to a first label 112. The second reagent 104 is a conjugate of a second antibody (or fragment thereof) 114 that specifically binds to the target analyte, conjugated to a second label 116. The third reagent 106 comprises a solid support 120 (such as, but not limited to, a magnetic latex particle) that is coated with streptavidin 122; the third reagent 106 further comprises a biotinylated third antibody (or fragment thereof) 124 that is prebound to the solid support 120 via the streptavidin coating 122. The third antibody 124 specifically binds to the second label 104 of the second reagent 104.Atty. 2024P19111US

[0089] As shown in FIG. 3, the antibodies 110 and 114 bind to substantially non-overlapping epitopes of the target analyte so that a sandwich complex 130 is formed. Then the sandwich complex formed of a target analyte having first and second reagents 102 and 104 bound thereto is captured on the third reagent 106 by binding of the antibody 124 to the second label 112.

[0090] FIG. 4 illustrates a specific (but non-limiting) embodiment of the present disclosure, in which the new immunoassay architecture disclosed in FIGS. 2-3 is applied to produce a new HBsAgll assay that mitigates any biotin interference observed in the commercial HBsAgll assay of FIG. 1. Like the commercial HBsAgll assay, the new assay 200 of FIG.4 is a sandwich immunoassay using direct, chemiluminometric technology. However, in the new assay 200, the reagents include an ancillary pack reagent 202 that comprises multiple fluorescein-labeled anti-HBs mouse monoclonal capture antibodies (indicated by reference numerals 204, 206, and 208, for purposes of example only) and an acridinium-ester-labeled anti-HBs mouse monoclonal antibody 210. Incubation with a biological sample allows HBsAg present in the sample (such as, but not limited to, AD / AY subtypes 212 and mutant HBsAg 214) to complex with two or more of the various antibodies 204, 206, 208, and 210 to form one or more complexes (indicated by reference numerals 220 and 222, for purposes of example only). A primary reagent pack 230 includes a solid reagent 232 and may further include a light reagent / second acridinium-ester-labeled anti-HBs mouse monoclonal antibody 234 (to increase the signal generated). The solid reagent 232 comprises a magnetic latex particle 236 that is coated with streptavidin 238, and also comprises a biotinylated antibody (or fragment thereof) 240 that is prebound to the magnetic latex particle 236 via the streptavidin coating 238 during the manufacturing process. The antibody 240 specifically binds to the fluorescein conjugated to the antibodies 204, 206, and 208.

[0091] The solid reagent 232 captures the sandwich complexes 220 and / or 222 via binding of the antibody 240 of the solid reagent 232 to the fluorescein bound to one or more of the antibodies 204, 206, and / or 208 to form one or more solid support-containing complexes 250, and a signal is generated by the acridinium esters of the conjugates 220 and 222 indirectly bound to the solid reagent 232. A direct relationship exists between the amount of HBsAg activity present in the patient sample and the amount of relative light units (RLUs) detected by the system.Atty. 2024P19111US

[0092] Table 1 illustrates a comparison of the new HBsAgll immunoassay of FIG. 4 to the commercially available existing HBsAgll immunoassay of FIG. 1 utilizing the ADVIA CENTAUR® XP system (Siemens Healthineers USA, Malvern, PA). As can be seen, when 3600 ng / ml biotin was present in the sample tested, the biotin bound to the streptavidin coated on the particles 52 and thus resulted in a non-reactive assay. However, the presence of 3600 ng / ml biotin did not interfere with the new HBsAgll assay, and both lowpositive and midpositive samples tested as reactive. The use of the preformed solid phase reagent positively impacted sensitivity and specificity of the assay.TABLE 1New HBsAgll Commercial HBsAgll(Biotin Mitigation) Lot 262 DTL1 R Sample ID Dose Interpretation Dose Interpretation 6325321_Control 0.31 Nonreact ive <0.10 Nonreactive Negative6325321_Test <0.10 Nonreact ive <0.10 Nonreactive SampleBiotin @ 3600 ng / mL: % Bias N / A N / APanel 2_Control 1.53 Reactive 1.70 Reactive LowPanel 2_Test <0.10 Nonreact ive 1.58 Reactive PositiveBiotin @ 3600 ng / mL: % Bias -100% -7.0%Panel 7_Control 244.25 Reactive 238.38 Reactive MidPanel 7_Test 1.99 Reactive 234.66 Reactive PositiveBiotin @ 3600 ng / mL: % Bias -99.2% -1.6%

[0093] This biotin interference testing was repeated with additional lots of the new HBsAgll immunoassay on the ADVIA CENTAUR® XP and Atellica IM systems (Siemens Healthineers USA, Malvern, PA), and the data is presented in Tables 2-3. As can be seen, the positive impacts on the sensitivity and specificity of the assay were again observed in this additional testing.Atty. 2024P19111USTABLE 2: ADVIA Centaur XPNew HBsAgll (Biotin Mitigation)DTL2 DTL3 DTL4 SamplelD Replicate RLU Dose RLU Dose RLU? DoseSamples Spiked with Biotin 1 Neg-3600-Bio i 5 2,031 0.09 i 1,833 i 0.05 i 1,765 i 0.07 i Low-3600-Bio 1 5 8,518 3.88 i 8,187 1 3.78 i 8,065 i 3.93 i Mid-3600-Bio| 5 41,66125.00: 40,356 j 24.68 j 40,017 i 24.48 j Control Samples (No Biotin)Neg-NaOH j 5 2,083 0.07 i 1,892! 0.02 j 1,7340.05 j ’ Low-NaOH i 5 8,712 4.00 i 8,094 i 3.72 j 8,043 i 3.92 j i Mid-NaOH 1 5 42,92125.82 i 40,760 j 24.95 i 40,578 i 24.85 iNegative;, No Change No Change No Change Interpretation s Low Positive % Interference i-3.0% 1.6% 0.3%; Mid Positive j % Interference i -3.2% -1.1% -1.5%TABLE 3: Atellica IMNew HBsAgll (Biotin Mitigation)DTL2 DTL3 DTL4 SamplelD Replicate RLU Dose RLU Dose RLU i Dose ISamples Spiked with Biotin 1 Neg-3600-Bio i 5 3,179 0.04 2,870 i 0.08 i 2,524 i 0.10 i i Low-3600-Bio j 5 15,574 4.05 14,521 j 3.96 i 14,073? 4.00 i I Mid-3600-Bio 15 77,27125.47 i 73,372! 25.06 i 71,102 i 24.76 i Control Samples (No Biotin)1 Neg-NaOH | 5 3,171 0.04 i 2,924 \ 0.10 i 2,559 i 0.11 i | Low-NaOH j 5 15,543 4.04 j 14,607 j 3.99 j 14,037; 3.98 j > Mid-NaOH! 5 77,524 25.56 > 72,806 | 24.85 i 71,544 j 24.92 i Negative i |nt^p^ationNo change No chanSe NoChange i Low Positive % Interference \ 0.2% -0.8% 0.5% iMid Positive; % Interference \ -0.4% 0.8% -0.6%

[0094] FIGS. 5 and 6 illustrates two other non-limiting embodiments of immunoassay architectures constructed in accordance with the present disclosure. The immunoassay format 400 of FIG. 5 and the immunoassay format 500 of FIG. 6 each utilize two reagents that areAtty. 2024P19111USidentical to two of the three reagents of the immunoassay 100 of FIGS. 2-3 but replace one of the antibodies in the first or second reagent 102 or 104 with the target analyte or an analog / derivative thereof, whereby the immunoassays 400 and 500 assume a competitive format. In particular, each of the immunoassays 400 and 500 utilize the unique prebound solid reagent 406 / 506 that is identical to the third reagent 106 of FIGS. 2-3 (i.e., a solid support that is coated with streptavidin and has a biotinylated antibody prebound thereto); however, one of the antibodies 110 or 114 bound to the first or second reagents 102 or 104 of the immunoassay 100 is replaced with an analyte or an analog / derivative thereof, so that the three reagents of the immunoassay bind in the absence of target analyte in the sample and form a sandwich complex when analyte is not present. As such, the amount of signal generated by these immunoassays is inversely proportional to the amount of analyte present in a sample.

[0095] In FIG. 5, the assay architecture 400 includes a first reagent 402, a second reagent 404, and the prebound solid reagent 406 (also referred to herein as a prebound third reagent 406). The first reagent 402 is a conjugate of an analyte or analog thereof 410 conjugated to a first label 412. The second reagent 404 is identical to the second reagent 104 of the immunoassay 100 and includes a conjugate of an anti-analyte antibody (or fragment thereof) 414 that specifically binds to the target analyte, conjugated to a second label 416. The prebound third reagent 406 comprises a solid support 420 (such as, but not limited to, a magnetic latex particle) that is coated with streptavidin 422; the third reagent 406 further comprises a biotinylated antibody (or fragment thereof) 424 that is prebound to the solid support 420 via the streptavidin coating 422. The antibody 424 specifically binds to the second label 416 of the second reagent 404. As shown in the upper panel of FIG. 5, when analyte is not present, the antibody 414 of the second reagent 404 binds to the analyte / analog 410 of the first reagent 402, and the antibody 424 of the third reagent 406 binds to the second label 412 of the first reagent 402 to thereby form a sandwich complex 430. However, when analyte is present as shown in the lower panel, the analyte from the sample competes with the first reagent 402 in binding to the second reagent 404, thereby reducing the amount of sandwich complexes 430 that are formed. As such, the amount of signal generated by the label 416 bound in the complex 430 is inversely proportional to the amount of target analyte present in the sample.Atty. 2024P19111US

[0096] The immunoassay 500 of FIG. 6 utilizes a first and third reagents 502 and 506 that are identical to the first and third reagents 102 and 106 of the immunoassay 100 of FIGS. 2-3. The immunoassay 500 differs in that a second reagent 504 includes a target analyte or analog thereof 510 that has a detectable label 512 bound thereto. As in the immunoassay 400, when analyte is not present, the three reagents 102, 104, and 106 form a sandwich complex 530 (upper panel of FIG. 6). However, when analyte is present, the analyte from the sample competes with the second reagent 504 for binding to the analyte-specific antibody of the first reagent 502, thereby reducing the amount of sandwich complexes 530 that are formed (lower panel of FIG. 6). As such, the amount of signal generated by the label 512 bound in the complex 530 is inversely proportional to the amount of target analyte present in the sample.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0097] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0098] Illustrative embodiment 1. A kit for performing an immunoassay for a target analyte, the kit comprising: a first reagent comprising a conjugate of a first label and a first antibody or antigen-binding fragment thereof that specifically binds to the target analyte; a second reagent comprising a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte; and a third reagent comprising a streptavidin-coated solid support and a third antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the third antibody or antigen-binding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigen-binding fragment thereof to the solid support; wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte; and wherein the first and second reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.Atty. 2024P19111US

[0099] Illustrative embodiment 2. The kit of illustrative embodiment 1, wherein the first label is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0100] Illustrative embodiment 3. The kit of illustrative embodiment 2, wherein the first label is an acridinium ester.

[0101] Illustrative embodiment 4. The kit of any of illustrative embodiments 1-3, wherein the second label is selected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0102] Illustrative embodiment 5. The kit of any of illustrative embodiments 1-4, wherein the second label is fluorescein, and wherein the third antibody or antigen-binding fragment thereof conjugated to the solid support is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0103] Illustrative embodiment 6. The kit of any of illustrative embodiments 1-5, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0104] Illustrative embodiment 7. The kit of any of illustrative embodiments 1-6, wherein the solid support comprises magnetic particles.

[0105] Illustrative embodiment 8. The kit of any of illustrative embodiments 1-7, wherein the solid support comprises magnetic latex particles.

[0106] Illustrative embodiment 9. The kit of any of illustrative embodiments 1-8, wherein the target analyte is selected from the group consisting of Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM); Hepatitis A virus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; and N-terminal peptide (PIIINP).

[0107] Illustrative embodiment 9A. The kit of illustrative embodiment 9, wherein the target analyte comprises HBsAg.

[0108] Illustrative embodiment 10. The kit of any of illustrative embodiments 1-9, further comprising at least one of: a fourth reagent that comprises a conjugate of the second label and a fourth antibody or antigen-binding fragment thereof that specifically binds to the targetAtty. 2024P19111USanalyte, wherein at least the first and fourth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the first and fourth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the fourth reagent to attach the sandwich composition to the solid support; and / or a fifth reagent that comprises a conjugate of the first label and a fifth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the second and fifth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the second and fifth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.

[0109] Illustrative embodiment 11. A kit for performing an immunoassay for a target analyte, the kit comprising: a first reagent comprising a conjugate formed of a first antibody or antigen-binding fragment thereof that specifically binds to the target analyte conjugated to an acridinium ester; a second reagent comprising a conjugate of a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte having a fluorescein conjugated thereto; and a third reagent comprising a streptavidin-coated solid support and an anti-fluorescein antibody or antigen-binding fragment thereof, wherein the anti-fluorescein antibody or antigen-binding fragment thereof is biotinylated such that the biotin is bound to the streptavidin on the solid support, thereby attaching the anti-fluorescein antibody or antigen-binding fragment thereof to the solid support; wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte; and wherein the first and second reagents bind to the same target analyte molecule to form a sandwich composition, and the anti-fluorescein antibody or antigen-binding fragment thereof of the third reagent binds to the fluorescein of the second reagent to attach the sandwich composition to the solid support.

[0110] Illustrative embodiment 12. The kit of illustrative embodiments 11, wherein the fluorescein is fluorescein isothiocyanate (FITC).Atty. 2024P19111US

[0111] Illustrative embodiment 13. The kit of illustrative embodiment 11 or 12, wherein the solid support comprises magnetic particles.

[0112] Illustrative embodiment 14. The kit of any of illustrative embodiments 11-13, wherein the solid support comprises magnetic latex particles.

[0113] Illustrative embodiment 15. The kit of any of illustrative embodiments 11-14, wherein the target analyte is selected from the group consisting of Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM); Hepatitis A virus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; and N-terminal peptide (PIIINP).

[0114] Illustrative embodiment 15A. The kit of illustrative embodiment 15, wherein the target analyte comprises HBsAg.

[0115] Illustrative embodiment 16. The kit of any of illustrative embodiments 11-15, further comprising at least one of: a fourth reagent that comprises a conjugate of the second label and a fourth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the first and fourth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the first and fourth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the fourth reagent to attach the sandwich composition to the solid support; and / or a fifth reagent that comprises a conjugate of the first label and a fifth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the second and fifth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the second and fifth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragmentAtty. 2024P19111USthereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.

[0116] Illustrative embodiment 17. A system for performing an immunoassay for a target analyte, the system comprising: the kit of any of illustrative embodiments 1-16; and a sample collection tube.

[0117] Illustrative embodiment 18. The system of illustrative embodiment 17, wherein the sample collection tube is a serum separator tube.

[0118] Illustrative embodiment 19. The system of illustrative embodiment 17 or 18, further comprising at least one additional reagent for initiating detection of the first label.

[0119] Illustrative embodiment 20. The system of illustrative embodiment 19, wherein the first label comprises an acridinium ester, and wherein the at least one additional reagent initiates detection of chemiluminescence triggered by the acridinium ester.

[0120] Illustrative embodiment 21. The system of illustrative embodiment 20, wherein the at least one additional reagent comprises an acid and a base.

[0121] Illustrative embodiment 22. A method of determining a concentration of a target analyte 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 conjugate of a first label and a first antibody or antigenbinding fragment thereof that specifically binds to the target analyte; (c) a second reagent comprising a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte; and (d) a third reagent comprising a streptavidin-coated solid support having a third antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the third antibody or antigenbinding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigen-binding fragment thereof to the solid support, and wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte; (2) incubating the mixture under conditions whereby (b) and (c) bind to target analyte present in the biological sample to form a sandwich composition, and the third antibody of (d) binds to the second label of (c) to attach theAtty. 2024P19111USsandwich composition to the solid support of (d); (3) detecting a signal generated by the first label of (b) on the solid support of (d); and (4) determining a concentration of the target analyte present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the first label on the solid support.

[0122] Illustrative embodiment 23. The method of illustrative embodiment 22, wherein the biological sample is at least one of blood, serum, or plasma.

[0123] Illustrative embodiment 24. The method of illustrative embodiment 22 or 23, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

[0124] Illustrative embodiment 25. The method of illustrative embodiment 24, wherein the sample collection tube is a serum separator tube.

[0125] Illustrative embodiment 26. The method of any of illustrative embodiments 22-25, further comprising performing a wash step prior to step (3).

[0126] Illustrative embodiment 27. The method of any of illustrative embodiments 22-26, wherein the first label of (b) is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0127] Illustrative embodiment 28. The method of any of illustrative embodiments 22-27, wherein the first label of (b) is an acridinium ester, and wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0128] Illustrative embodiment 29. The method of any of illustrative embodiments 22-28, wherein the second label of (c) is selected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0129] Illustrative embodiment 30. The method of any of illustrative embodiments 22-29, wherein the second label of (c) is fluorescein.

[0130] Illustrative embodiment 31. The method of illustrative embodiment 30, wherein the third antibody or antigen-binding fragment thereof conjugated to the solid support of (d) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.Atty. 2024P19111US

[0131] Illustrative embodiment 32. The method of any of illustrative embodiments 22-31, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0132] Illustrative embodiment 33. The method of any of illustrative embodiments 22-32, wherein the solid support of (d) comprises magnetic particles.

[0133] Illustrative embodiment 34. The method of any of illustrative embodiments 22-33, wherein the solid support comprises magnetic latex particles.

[0134] Illustrative embodiment 35. The method of any of illustrative embodiments 22-34, wherein the target analyte is selected from the group consisting of Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM); Hepatitis A virus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; and N-terminal peptide (PIIINP).

[0135] Illustrative embodiment 35A. The method of illustrative embodiment 35, wherein the target analyte comprises HBsAg.

[0136] Illustrative embodiment 36. The method of any of illustrative embodiments 22-35, wherein in step (1), (c) and (d) are combined prior to addition of (a).

[0137] Illustrative embodiment 37. The method of any of illustrative embodiment 22-36, wherein step (1) further comprises the addition of at least one of (d) or (e) to form the mixture: (d) a fourth reagent that comprises a conjugate of the second label and a fourth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the first and fourth antibodies or antigen-binding fragments thereof bind to substantially nonoverlapping epitopes of the target analyte such that the first and fourth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigenbinding fragment thereof of the third reagent binds to the second label of the fourth reagent to attach the sandwich composition to the solid support; and / or (e) a fifth reagent that comprises a conjugate of the first label and a fifth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the second and fifth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the second and fifth reagents bind to the same target analyte molecule to formAtty. 2024P19111USa sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.

[0138] Illustrative embodiment 38. The method of illustrative embodiment 37, wherein when (d) is added: in step (2), a first sandwich composition comprising (b) and (c) is formed and attached to the solid support of (d), and a second sandwich composition comprising (b) and (d) is formed and attached to the solid support of (d); and wherein the signal generated by the first label of (b) in both the first and second sandwich compositions on the solid support of (d) is detected in step (3).

[0139] Illustrative embodiment 39. The method of illustrative embodiment 37 or 38, wherein when (e) is added: in step (2), a first sandwich composition comprising (b) and (c) is formed and attached to the solid support of (d), and a second sandwich composition comprising (e) and (c) is formed and attached to the solid support of (d); and wherein the signal generated by the first labels of (b) and (e) in both the first and second sandwich compositions on the solid support of (d) is detected in step (3).

[0140] Illustrative embodiment 40. A kit for performing a competitive immunoassay for a target analyte, the kit comprising: a first reagent comprising a conjugate of a first label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte; a second reagent comprising a conjugate of a second label and a target analyte or analog thereof to which the antibody or antigen-binding fragment thereof can specifically bind; and a third reagent comprising a streptavidin-coated solid support and an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the antibody or antigenbinding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support.

[0141] Illustrative embodiment 41. A kit for performing a competitive immunoassay for a target analyte, the kit comprising: a first reagent comprising a conjugate of a first label and a target analyte or analog thereof; a second reagent comprising a conjugate of a second label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte orAtty. 2024P19111USanalog thereof; and a third reagent comprising a streptavidin-coated solid support and an antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigenbinding fragment thereof to the solid support.

[0142] Illustrative embodiment 42. The kit of illustrative embodiment 40 or 41, 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.

[0143] Illustrative embodiment 43. The kit of illustrative embodiment 42, wherein the first label is an acridinium ester.

[0144] Illustrative embodiment 44. The kit of any of illustrative embodiments 41-43, wherein the second label is selected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0145] Illustrative embodiment 45. The kit of any of illustrative embodiments 41-44, wherein the second label is fluorescein, and wherein the antibody or antigen-binding fragment thereof conjugated to the solid support in the third reagent is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0146] Illustrative embodiment 46. The kit of any of illustrative embodiments 41-45, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0147] Illustrative embodiment 47. The kit of any of illustrative embodiments 41-46, wherein the solid support comprises magnetic particles.

[0148] Illustrative embodiment 48. The kit of any of illustrative embodiments 41-47, wherein the solid support comprises magnetic latex particles.

[0149] Illustrative embodiment 49. The kit of any of illustrative embodiments 41-48, wherein the target analyte is selected from the group consisting of Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM); Hepatitis A virus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; and N-terminal peptide (PIIINP).Atty. 2024P19111US

[0150] Illustrative embodiment 49A. The kit of illustrative embodiment 49, wherein the target analyte comprises HBsAg.

[0151] Illustrative embodiment 50. A system for performing an immunoassay for a target analyte, the system comprising: the kit of any of illustrative embodiments 41-49; and a sample collection tube.

[0152] Illustrative embodiment 51. The system of illustrative embodiment 50, wherein the sample collection tube is a serum separator tube.

[0153] Illustrative embodiment 52. The system of illustrative embodiment 50 or 51, further comprising at least one additional reagent for initiating detection of the first label.

[0154] Illustrative embodiment 53. The system of illustrative embodiment 52, wherein the first label comprises an acridinium ester, and wherein the at least one additional reagent initiates detection of chemiluminescence triggered by the acridinium ester.

[0155] Illustrative embodiment 54. The system of illustrative embodiment 53, wherein the at least one additional reagent comprises an acid and a base.

[0156] Illustrative embodiment 55. A method of determining a concentration of a target analyte in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a conjugate of a first label and an antibody or antigenbinding fragment thereof that specifically binds to the target analyte; (c) a second reagent comprising a conjugate of a second label and a target analyte or analog thereof to which the antibody or antigen-binding fragment of (b) can specifically bind; and (d) a third reagent comprising a streptavidin-coated solid support having an antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support; (2) incubating the mixture under conditions whereby, in the absence of target analyte present in the biological sample, (b) and (c) bind to form a sandwich composition, and the antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d), and whereby in the presence of target analyte in theAtty. 2024P19111USbiological sample, (b) binds to the target analyte, and the sandwich composition does not form; (3) detecting a signal generated by the first label of (b) on the solid support of (d); and (4) determining a concentration of the target analyte 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.

[0157] Illustrative embodiment 56. A method of determining a concentration of a target analyte in a biological sample, the method comprising the steps of: (1) combining, either simultaneously or wholly or partially sequentially, (a)-(d) to form a mixture: (a) a biological sample; (b) a first reagent comprising a conjugate of a first label and a target analyte or analog thereof; (c) a second reagent comprising a conjugate of a second label and an antibody or antigen-binding fragment thereof that specifically binds to the target analyte or analog thereof; and (d) a third reagent comprising a streptavidin-coated solid support having an antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the antibody or antigen-binding fragment thereof of the third reagent is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the antibody or antigen-binding fragment thereof to the solid support; (2) incubating the mixture under conditions whereby, in the absence of target analyte present in the biological sample, (b) and (c) bind to form a sandwich composition, and the antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d), and whereby in the presence of target analyte in the biological sample, (c) binds to the target analyte, and the sandwich composition does not form; (3) detecting a signal generated by the first label of (b) on the solid support of (d); and (4) determining a concentration of the target analyte 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.

[0158] Illustrative embodiment 57. The method of illustrative embodiment 55 or 56, wherein the biological sample is at least one of blood, serum, or plasma.

[0159] Illustrative embodiment 58. The method of any of illustrative embodiments 55-57, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.Atty. 2024P19111US

[0160] Illustrative embodiment 59. The method of illustrative embodiment 58, wherein the sample collection tube is a serum separator tube.

[0161] Illustrative embodiment 60. The method of any of illustrative embodiments 55-59, further comprising performing a wash step prior to step (3).

[0162] Illustrative embodiment 61. The method of any of illustrative embodiments 55-60, wherein the first label of (b) is selected from the group consisting of a chemiluminescent compound, a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, and an enzyme.

[0163] Illustrative embodiment 62. The method of any of illustrative embodiments 55-61, wherein the first label of (b) is an acridinium ester, and wherein step (3) comprises adding at least one additional reagent that triggers chemiluminescence that is quantified as relative light units (RLUs).

[0164] Illustrative embodiment 63. The method of any of illustrative embodiments 55-62, wherein the second label of (c) is selected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.

[0165] Illustrative embodiment 64. The method of any of illustrative embodiments 55-63, wherein the second label of (c) is fluorescein.

[0166] Illustrative embodiment 65. The method of illustrative embodiment 64, wherein the third antibody or antigen-binding fragment thereof conjugated to the solid support of (d) is an anti-fluorescein monoclonal antibody or antigen-binding fragment thereof.

[0167] Illustrative embodiment 66. The method of any of illustrative embodiments 55-65, wherein the fluorescein is fluorescein isothiocyanate (FITC).

[0168] Illustrative embodiment 67. The method of any of illustrative embodiments 55-66, wherein the solid support of (d) comprises magnetic particles.

[0169] Illustrative embodiment 68. The method of any of illustrative embodiments 55-67, wherein the solid support comprises magnetic latex particles.

[0170] Illustrative embodiment 69. The method of any of illustrative embodiments 55-68, wherein the target analyte is selected from the group consisting of Hepatitis B surface antigen (HBsAg); Testosterone II (TSTII); Folate (FOL); Hepatitis A virus (HAV) IgM antibody (aHAVM);Atty. 2024P19111USHepatitis Avirus total antibodies (aHAVT / HAVT); Hepatitis B core IgM antibody (aHBcM); DHEAS-SO4(dehydroepiandrosterone sulfate); sex hormone-binding globulin (SHBG); cyclosporine (CsA); Procollagen III; and N-terminal peptide (PIIINP).

[0171] Illustrative embodiment 69A. The method of illustrative embodiment 69, wherein the target analyte comprises HBsAg.

[0172] Illustrative embodiment 70. The method of any of illustrative embodiments 55-69, wherein in step (1), (c) and (d) are combined prior to addition of (a).

[0173] 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

Atty. 2024P19111USCLAIMS1. A kit for performing an immunoassay for a target analyte, the kit comprising:a first reagent comprising a conjugate of a first label and a first antibody or antigenbinding fragment thereof that specifically binds to the target analyte;a second reagent comprising a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte; and a third reagent comprising:a streptavidin-coated solid support;a third antibody or antigen-binding fragment thereof that specifically binds to the second label, wherein the third antibody or antigen-binding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigen-binding fragment thereof to the solid support;wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte; and wherein the first and second reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.

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

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

4. The kit of claim 1, wherein the second label is selected from the group consisting of fluorescein, streptavidin, an anti-biotin antibody or antigen-binding fragment thereof, and digoxigenin.Atty. 2024P19111US5. The kit of claim 4, wherein the second label is a fluorescein, and wherein the third antibody or antigen-binding fragment thereof conjugated to the solid support is an antifluorescein monoclonal antibody or antigen-binding fragment thereof.

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

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

8. The kit of claim 1, wherein the target analyte comprises hepatitis B surface antigen (HBsAg).

9. The kit of claim 1, further comprising at least one of:a fourth reagent that comprises a conjugate of the second label and a fourth antibody or antigen-binding fragment thereof that specifically binds to the target analyte, wherein at least the first and fourth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the first and fourth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the fourth reagent to attach the sandwich composition to the solid support; and / ora fifth reagent that comprises a conjugate of the first label and a fifth antibody or antigenbinding fragment thereof that specifically binds to the target analyte, wherein at least the second and fifth antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte such that the second and fifth reagents bind to the same target analyte molecule to form a sandwich composition, and the third antibody or antigen-binding fragment thereof of the third reagent binds to the second label of the second reagent to attach the sandwich composition to the solid support.Atty. 2024P19111US10. A system for performing an immunoassay for a target analyte, the system comprising:the kit of any of claims 1-9; anda sample collection tube.

11. The system of claim 10, wherein the sample collection tube is a serum separator tube.

12. The system of claim 10, further comprising at least one additional reagent for initiating detection of the first label.

13. A method of determining a concentration of a target analyte 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 conjugate of a first label and a first antibody or antigen-binding fragment thereof that specifically binds to the target analyte;(c) a second reagent comprising a conjugate of a second label and a second antibody or antigen-binding fragment thereof that specifically binds to the target analyte; and(d) a third reagent comprising a streptavidin-coated solid support having a third antibody or antigen-binding fragment thereof that specifically binds to the second label bound thereto, wherein the third antibody or antigenbinding fragment thereof is biotinylated such that the biotin binds to the streptavidin on the solid support and attaches the third antibody or antigen-binding fragment thereof to the solid support, and wherein the first and second antibodies or antigen-binding fragments thereof bind to substantially non-overlapping epitopes of the target analyte;Atty. 2024P19111US(2) incubating the mixture under conditions whereby (b) and (c) bind to target analyte present in the biological sample to form a sandwich composition, and the third antibody of (d) binds to the second label of (c) to attach the sandwich composition to the solid support of (d);(3) detecting a signal generated by the first label of (b) on the solid support of (d); and (4) determining a concentration of the target analyte present in the biological sample, wherein the concentration is proportional to the amount of signal generated by the first label on the solid support.

14. The method of claim 13, wherein the biological sample is at least one of blood, serum, or plasma.

15. The method of claim 14, further comprising the step of performing a separation of the biological sample prior to step (1) using at least one sample collection tube.

16. The method of claim 13, further comprising performing a wash step prior to step (3).

17. The method of claim 13, wherein the first label of (b) 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).

18. The method of claim 13, wherein the second label of (c) is fluorescein, and the third antibody or antigen-binding fragment thereof bound to the solid support of (d) is an antifluorescein monoclonal antibody or antigen-binding fragment thereof.

19. The method of claim 13, wherein the solid support of (c) comprises magnetic particles.

20. The method of claim 13, wherein the target analyte is hepatitis B surface antigen (HBsAg).

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