Reagent compositions for immunoassays and their use in the detection of cancer antigen 19-9.

Abstract

This document provides reagents for an immunoassay to detect cancer antigen 19-9 (CA 19-9), which contain a divalent salt, such as MgSO4. Kits comprising said reagents are also provided, as well as methods and systems for detecting CA 19-9 in samples, such as plasma or serum samples.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 595,388, filed November 2, 2023. The entire disclosure of the above application is incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to methods, compositions, kits, and systems for detecting the presence and / or amount of cancer antigen 19-9 (CA 19-9) in biological samples. Specifically, this disclosure relates to reagents, such as solid-phase reagent compositions comprising divalent salts (e.g., MgSO4) and solid phases having anti-CA 19-9 monoclonal antibodies or fragments thereof directly or indirectly attached thereto, and the use of such reagents for detecting CA 19-9. Background Technology

[0003] This section provides background information relating to this disclosure, which is not necessarily prior art.

[0004] Pancreatic cancer is difficult to diagnose early due to the lack of physical signs or symptoms. By the time symptoms begin to appear, the tumor has often grown very large or spread outside the pancreas. Therefore, pancreatic cancer has one of the lowest survival rates of all cancers.

[0005] Tumor markers are used in the diagnosis and management of various cancers. In particular (but not limitingly), cancer antigen 19-9 (CA 19-9) is a tumor marker for pancreatic and colorectal cancers. CA 19-9 is a glycoprotein, a sialylated form of the Lewis blood group antigen, and exists in serum as a mucin. CA 19-9 is synthesized by normal human pancreatic and bile duct cells, as well as by the gastric, colonic, endometrial, and salivary epithelium, and elevated serum CA 19-9 levels can be an indicator of pancreatic, gastric, and hepatobiliary malignancies. The first monoclonal antibodies against CA 19-9 were developed from human colon cancer cell lines, as disclosed in 1979 (Koprowski et al. (1979)). Somatic Cell Genet (5:957-971), and since then, various immunoassays of CA19-9 have been developed and are commercially available. The performance characteristics of these commercially available CA 19-9 assays have also been evaluated (see, for example (but not limited to) Stern et al. (2001)). Clin Chem Lab Med ., 39:1278-1282; and La'ulu et al. (2007) Am J Clin Pathol(127:436-440). Non-limiting examples of commercially available CA 19-9 assays include the ADVIA® Centaur CA 19-9 assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY); the Atellaca IM® CA 19-9 assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY); the ARCHITECT® / 2000 CA 19-9XR assay (Abbott Diagnostics, Abbott Park, IL); the IMMULITE® 2000 GI-MA assay (Diagnostic Products, Los Angeles, CA); the Elecsys E170 CA 19-9 assay (Roche Diagnostics, Indianapolis, IN); and the UniCel® Dxl800 GL Monitor (Beckman Coulter, Fullerton, CA).

[0006] Despite the availability of commercially available immunoassays, the sensitivity and accuracy observed for these assays require improvement. For example, it should be demonstrated that CA 19-9 recoveries are substantially equivalent across different sample types when using various forms of biological samples (such as serum, EDTA plasma, lithium heparin plasma, etc.). However, it has been found that the matrices of various biological samples often differ and can interfere with the accuracy of the assays.

[0007] Therefore, there is a need in the art for novel and improved reagent compositions that overcome the shortcomings of existing assays for use in immunoassays. This disclosure relates to such reagent compositions, kits thereof, and methods of use. Summary of the Invention

[0008] This section provides a general overview of this disclosure and is not a full disclosure of its entire scope or all its features.

[0009] In some aspects, this disclosure provides a solid-phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9). The solid-phase reagent composition comprises a solid phase having an anti-CA 19-9 monoclonal antibody or a fragment thereof directly or indirectly attached thereto, and a divalent salt, such as MgSO4.

[0010] In other aspects, this disclosure provides a liquid phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9). The liquid phase reagent composition comprises a conjugate and a divalent salt, such as MgSO4. The conjugate comprises an anti-CA 19-9 monoclonal antibody or a fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups, and at least one label is directly or indirectly attached to the two thiol groups of the anti-CA 19-9 monoclonal antibody or the fragment thereof, thereby forming the conjugate; wherein the label is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I. Formula I.

[0011] In other aspects, this disclosure provides an immunoassay kit for detecting cancer antigen 19-9 (CA 19-9), comprising a solid-phase reagent and a liquid-phase reagent. The solid-phase reagent comprises a solid phase having an anti-CA 19-9 monoclonal antibody or a fragment thereof directly or indirectly attached thereto, and a divalent salt, such as MgSO4. The liquid-phase reagent comprises a conjugate and optionally a divalent salt, such as MgSO4. The conjugate comprises an anti-CA 19-9 monoclonal antibody or a fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups, and at least one label is directly or indirectly attached to the two thiol groups of the anti-CA 19-9 monoclonal antibody or the fragment thereof, thereby forming the conjugate; wherein the label is DiBrPD-HEGAE having the structure of Formula I.

[0012] In other aspects, this disclosure provides a method for detecting cancer antigen 19-9 (CA 19-9) in a sample. The method includes: combining a sample with a solid-phase reagent composition and a liquid-phase reagent composition of an immunoassay kit described herein to form a mixture; incubating the mixture under conditions that allow the solid-phase reagent composition and the liquid-phase reagent composition to bind to CA 19-9 present in the sample, thereby forming a complex; and detecting the complex.

[0013] In other aspects, this disclosure provides a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the method for detecting cancer antigen 19-9 (CA 19-9) in a sample as described herein.

[0014] Other applicable areas will become apparent from the description provided herein. The descriptions and specific examples in this overview are for illustrative purposes only and are not intended to limit the scope of this disclosure. Attached Figure Description

[0015] The accompanying drawings described herein are for illustrative purposes only and do not represent all possible embodiments, nor are they intended to limit the scope of this disclosure.

[0016] Figure 1 A non-limiting embodiment of an immunoassay for detecting cancer antigen CA 19-9 according to Example 1 of this disclosure is illustrated. The immunoassay employs a solid-phase reagent comprising a divalent salt and paramagnetic particles (PMPs) covalently labeled with an anti-CA 19-9 monoclonal antibody, and a liquid-phase reagent comprising an anti-CA 19-9 monoclonal antibody-acrididine ester (CA19-9-MAb-AE) conjugate.

[0017] Figure 2 This illustration depicts a non-limiting embodiment of the immunoassay for detecting cancer antigen CA 19-9 according to Example 2 of this disclosure. The immunoassay employs a solid-phase reagent comprising a divalent salt and paramagnetic particles (PMPs) covalently labeled with anti-CA 19-9 monoclonal antibody, and a liquid-phase reagent comprising an anti-CA 19-9 monoclonal antibody-acrididine ester (CA19-9-MAb-AE) conjugate.

[0018] Figure 3 The graph shows the CA 19-9 measurements from K2 EDTA plasma versus the CA 19-9 measurements from matched samples, as determined in Example 2.

[0019] Figure 4 The graph shows the CA 19-9 measurements from K3 EDTA plasma versus the CA 19-9 measurements from matched serum samples, as determined in Example 2.

[0020] Figure 5 An exemplary block diagram is provided to illustrate a computer system suitable for performing the methods described in this disclosure on a chemical analyzer. Invention Details Exemplary embodiments are provided to make this disclosure thorough and complete, and to fully convey the scope of protection to those skilled in the art. Numerous specific details, such as examples of specific compositions, components, apparatuses, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that specific details are not required, exemplary embodiments may be embodied in many different forms, and none should be construed as limiting the scope of this disclosure. In some exemplary embodiments, well-known processes, well-known apparatus structures, and well-known technologies are not described in detail.

[0022] A. Definition The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having” are inclusive and therefore indicate the presence of said features, elements, compositions, steps, integers, operations, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof. Although the open-ended term “comprises” should be understood as a non-limiting term used to describe and claim the various embodiments set forth herein, in some respects it may be understood alternatively as a more restrictive and binding term, such as “consisting of” or “essentially composed of.” Therefore, for any given embodiment listing the compositions, materials, components, elements, features, integers, operations, and / or process steps, this disclosure also specifically includes embodiments consisting of, or substantially consisting of, the compositions, materials, components, elements, features, integers, operations, and / or process steps so listed. In the case of “consisting of…”, alternative embodiments exclude any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “substantially consisting of…”, such embodiments exclude any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that substantially affect the essential and novel features, but embodiments may include any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not substantially affect the essential and novel features.

[0023] Unless the order of execution is explicitly specified, no method, process, or operation described herein should be construed as requiring them to be performed in the specific order discussed or described. Unless otherwise stated, it should be understood that additional or alternative steps may be employed.

[0024] When used in conjunction with the term "comprising" in the claims and / or description, the term "a" or "an" may mean "one / a type," but it is also consistent with the meaning of "one or more / a plurality of," "at least one / a type," and "one / a type or more than one / a type." Therefore, unless the context clearly indicates otherwise, the terms "a," "an," and "the / described" include plural indicators. Thus, for example, reference to "compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plural" means "two or more."

[0025] The term "at least one" should be understood to include one and any number 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 it is attached to; furthermore, a quantity of 100 / 1000 is not considered limiting, as higher limits can also produce satisfactory results. Additionally, the term "at least one of X, Y, and Z" should be understood to include individual X, individual Y, and individual Z, as well as any combination of X, Y, and Z. For example, the use of ordinal numbers (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing two or more items and is not intended to imply any order or sequence of importance or any additional order of items.

[0026] The term "or" as used in the claims is intended to mean inclusive "and / or" unless explicitly stated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

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

[0028] Throughout this application, the term "about" is used to indicate that a value includes variations in the composition / instrument / apparatus, the inherent error of the method used to determine the value, or variations existing between the subjects under study. For example, but not as a limitation, when using the term "about," a specified value can vary from a specified value by positive or negative 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, such variations are suitable for performing the disclosed method and as understood by one of ordinary skill in the art.

[0029] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is significant in the particular context. Continuing this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so on. Those skilled in the art will understand that, unless obvious from the context, there is generally no limit to the number of items or terms in any combination.

[0030] As used herein, the term “substantially” means that the event or situation subsequently described occurred completely or to a great extent. For example, when relating to a particular event or situation, the term “substantially” means that the event or situation subsequently described occurred at least 80% of the time, or at least 85% of the time, at least 90% of the time, or at least 95% of the time.

[0031] Furthermore, the disclosure of the range includes the disclosure of all values ​​within the entire range and further subdivisions of the range, including the endpoints and subranges given for the range.

[0032] The term "conjugate" refers to any substance formed by two parts joined together. Representative conjugates according to this disclosure include those formed by linking a small molecule with a large molecule, such as a protein.

[0033] As used herein, the term "sample" should be understood to include any type of biological sample that may be utilized under this disclosure. Examples of usable fluid biological samples include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder irrigation fluid, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, etc.

[0034] The terms “specific binding partner” or “analyte-specific binder” should be understood to refer to any molecule capable of specifically binding to a target analyte. For example, but not limited to, binders / binding partners can be antibodies, receptors, ligands, aptamers, molecularly imprinted polymers (i.e., inorganic matrices), any fragments thereof, any combination or derivative thereof, and any other molecules capable of specifically binding to a target analyte.

[0035] The term “antibody (abbreviated as “Ab”)” is used in the broadest sense and specifically (but not limited to) encompasses monoclonal antibodies (abbreviated as “MAb”) (including full-length monoclonal antibodies), polyclonal antibodies (abbreviated as “pAb”), multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, provided they exhibit the desired biological activity of analyte binding. Therefore, the term “antibody” or “antibody peptide” refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or its antigen-binding fragment that competes with the intact antibody for specific antigen binding. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, biantibodies, single-chain antibodies, single-domain antibodies (e.g., but not limited to NANOBODIES®), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, for example, Hudson et al. (Nature Med. (2003) 9:129-134). Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). As used herein, the term “antigen-binding fragment” or “antigen-binding portion” of an antibody refers to one or more antibody fragments that retain the ability to bind antigens. The antigen-binding function of an antibody can be performed by fragments of the whole antibody. Examples of binding fragments covered 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, biantibodies, single-chain antibodies, single-domain antibodies (e.g., but not limited to NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of the whole antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as the whole antibody.

[0036] The term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, an epitope is an antigenic region that an antibody specifically binds to. Epitope determinants typically include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In some embodiments, an epitope may have specific three-dimensional structural properties (e.g., "conformational epitopes") and specific charge properties.

[0037] If a particular antibody specifically binds to two epitopes, the epitope is defined as "identical" to the other epitope. In some embodiments, polypeptides with different primary amino acid sequences can contain the same epitope. In some embodiments, the same epitope can have a different primary amino acid sequence. If different antibodies compete for specific binding to that epitope, they are referred to as binding to the same epitope.

[0038] When an antibody preferentially recognizes an antigen in a complex mixture of proteins and / or macromolecules, it "specifically binds" to that antigen. In some embodiments, the antibody contains an antigen-binding site that specifically binds to a particular epitope. In some such embodiments, the antibody is able to bind to different antigens, as long as the different antigens contain that particular epitope or closely related epitopes. In some cases, for example, homologous proteins from different species may contain the same epitope. In some embodiments, the antibody specifically binds to an antigen with a dissociation constant not greater than 10. -6 M, 10 -7 M, 10 -8 M or 10 -9 M. When an antibody specifically binds to a receptor or ligand (i.e., an anti-receptor), it can substantially inhibit receptor-ligand adhesion. As used herein, the antibody substantially inhibits receptor-ligand adhesion when an excess of antibody reduces the amount of receptor-ligand binding by at least about 20%, 40%, 60%, or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0039] "Isolated" antibodies are antibodies that have been separated from and / or recovered from components in the environment in which they were produced. Contaminant components in the production environment are materials that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody is purified to: 1) greater than 50% by weight, e.g., more than 75%, 85%, 95%, or 99% by weight, as determined by the Lowry method; 2) to the extent that a sequence of at least 10 N-terminal or internal amino acid residues, e.g., at least 15 residues, is obtained using a spin-cup sequencer; or 3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or alternative silver staining. Isolated antibodies include in situ antibodies within recombinant cells because at least one component in the environment from which the antibody was produced will be absent. However, isolated antibodies are typically prepared through at least one purification step. Furthermore, "isolated antibodies" generally do not contain other antibodies with different antigen specificities. However, isolated antibodies may exhibit some cross-reactivity with other related antigens.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies that specifically bind to the same epitope; that is, the individual antibodies constituting the group are identical except for the possibility of naturally occurring mutations present in small amounts. In contrast to conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that, in a production method, they can be synthesized from hybridoma cultures and are therefore free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody as being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, in one embodiment, the monoclonal antibody produced according to this disclosure can be prepared by a hybridoma method first described by Kohler and Milstein (Nature, 256: 495 (1975)).

[0041] The monoclonal antibodies utilized according to this disclosure can be produced by any method known in the art, including but not limited to the result of an intentional immunization protocol; the result of an immune response leading to the natural production of antibodies during a disease or cancer process; phage-derived antibodies; and so on. In addition to the hybridoma production methods listed above, the monoclonal antibodies of this disclosure can be produced by a variety of other methods, such as, but not limited to, recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567); isolating antibody fragments from phage display libraries (see, for example, Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); and various other monoclonal antibody production techniques (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold SpringHarbor Laboratory, Cold Spring Harbor, NY)). Furthermore, many of the monoclonal antibodies that can be used in the conjugates and methods disclosed herein or otherwise conceived are widely commercially available, and therefore further description thereof is deemed unnecessary.

[0042] An analyte is a molecule that can be recognized by an analyte-specific binding partner, such as (but not limited to) an antibody. An analyte contains at least one antigenic determinant or "epitope," which is the analyte region that binds to an analyte-specific binding partner (i.e., an antibody).

[0043] For example (but not limited to), the assay may include (i) a composition comprising: a chemiluminescent compound that is activatable by singlet oxygen and a fluorescent molecule excited by the activated chemiluminescent compound; and (ii) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state.

[0044] The term "peptide" refers to any compound formed by linking two or more amino acids via amide bonds. Representative peptides include polymers of α-amino acids, where the α-amino group of each non-terminal amino acid residue is linked to the α-carboxyl group of an adjacent residue in the linear chain. High molecular weight peptides are called "proteins."

[0045] The term "label" refers to a member of a signal generation system. Labels can be detected directly or through a specific binding reaction that generates a detectable signal. For example, a label can be an identification tag that attaches to a carrier substance or molecule to detect an analyte. Labels are typically radioactive isotopes, luminescent materials, particulate matter, or enzymes. Labels can be poly(amino acids), proteins, or non-poly(amino acids), isotopes or non-isotopes, usually non-isotopes, and can be catalysts, such as enzymes (e.g., β-galactosidase, peroxidase, etc.), polynucleotides encoding catalysts, promoters, dyes, fluorescent molecules (e.g., rhodamine, fluorescein isothiocyanate, or FITC), chemiluminescent molecules (e.g., dioxane, fluorescein, etc.), coenzymes, enzyme substrates, radioactive groups (e.g., ... 125 I) Protein-binding couplers (such as biotin), small organic molecules, amplifiable polynucleotide sequences, particles (such as latex or carbon particles, metal sols, microcrystals, liposomes, cells, etc.), which may or may not be labeled with dyes, catalysts or other detectable groups.

[0046] The term "non-poly(amino acid) label" refers to labels that are not proteins (such as enzymes). Non-poly(amino acid) labels can be members of signaling systems.

[0047] The signal generation system may have one or more components, at least one of which is a label. The signal generation system generates a signal associated with the presence of entactogens in the sample. The signal generation system contains all the reagents necessary to generate a measurable signal. Other components of the signal generation system may be contained in the chromogenic solution and may include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required to bind the signal-generating substance, etc. Other components of the signal generation system may also be coenzymes, substances that react with enzyme products, other enzymes, and catalysts, etc. The signal generation system provides a signal that can be detected by external tools, by using electromagnetic radiation, ideally by visual observation. An exemplary signal generation system is described in U.S. Patent No. 5,508,178 (Rose et al.), the disclosure of which is incorporated herein by reference.

[0048] The phrase “detection of analyte” refers to any quantitative, semi-quantitative, or qualitative method, and more generally to all other methods used to determine analytes, and especially to all other methods of CA 19-9. The terms “detection,” “determination,” and “identification” are used synonymously herein and are all within the scope of this disclosure.

[0049] The phrase "kit" or the term "kit" refers to a set of materials used to perform an assay. Depending on their cross-reactivity and stability, the reagents provided in the package may be housed in the same or separate containers and are in liquid or lyophilized form. The amount and proportion of reagents provided in the kit can be selected to provide optimal results for a specific application. Kits embodying the features of this disclosure contain antibodies specifically against CA 19-9, their conjugates, and / or enzymes or proteins necessary for detecting the presence and amount of CA19-9 or its complexes in a sample. The kit may further include calibration and control materials. Reagents may be kept in liquid form or may be lyophilized.

[0050] The phrase "calibration and control materials" refers to any standard or reference material containing a known amount of the analyte. Samples suspected of containing the analyte and their corresponding calibration materials are measured under similar conditions. The analyte concentration is calculated by comparing the results obtained from the unknown sample with those obtained from the standard. This is typically done by plotting a calibration curve.

[0051] B. Reagent compositions for immunoassays This article provides compositions of immunoassay reagents for the detection of cancer antigen 19-9 (CA 19-9). As mentioned above, the matrix of different types of biological samples often varies and can interfere with the accuracy of the assay. For example, it has been found that in CA 19-9 assays, the recovery of CA 19-9 in plasma samples (such as EDTA plasma) is higher than in serum samples, resulting in undesirable EDTA plasma bias. To address this issue, reagent compositions containing divalent salts are provided, which advantageously mitigate EDTA plasma bias in plasma samples. Without being bound by theory, it is believed that the addition of divalent salts (such as magnesium sulfate (MgSO4)) can mitigate EDTA plasma bias because EDTA reacts well with divalent cations (such as Mg... 2+ These salts exhibit higher binding affinity. Therefore, this document provides reagent compositions comprising divalent salts, such as solid-phase and liquid-phase reagent compositions. It is envisioned that the provided reagent compositions are not limited to use in the determination of plasma samples, but are also suitable for the determination of various biological samples as described herein.

[0052] In any embodiment, the solid-phase reagent composition comprises a solid-phase reagent having an anti-CA 19-9 monoclonal antibody or a fragment thereof (also referred to as "CA 19-9 MAb" or "MAb 19-9") directly or indirectly attached thereto, and a divalent salt. Anti-CA19-9 monoclonal antibodies / fragments thereof are known in the art and commercially available; for example (but not limitingly), anti-CA19-9 monoclonal antibodies that can be used according to this disclosure are available from: Abeam (Cambridge, UK); Abnova Corporation (Walnut, CA); Agilent Technologies (Santa Clara, CA); Antibodies-Online Inc. (Limerick, PA); Biocare Medical (Pacheco, CA); Biobyt Ltd. (St. Louis, MO); Creative Diagnostics (Shirley, NY); Cell Sciences (Newburyport, MA); EastCoastBio (North Berwick, ME); Enzo Life Sciences, Inc. (Farmingdale, NY); Fitzgerald Industries International (Acton, MA); LifeSpan BioSciences (Seattle, WA); MyBioSource, Inc. (San Diego, CA); OriGene Technologies, Inc. (Rockville, MD); RayBiotech Life (Peachtree Corners, GA); Sigma Aldrich (St. Louis, MO); ThermoFisher Scientific (Waltham, MA); US Biological Life Sciences (Salem, MA) and many other suppliers. For example, anti-CA 19-9 MAb (catalog number: 210-580; clone 1116NS19-9) from Fujirebio Diagnostics Inc. (Malvern, PA), or any other anti-CA 19-9 monoclonal antibody / fragment disclosed above, can be used. The solid-phase reagent may contain any solid-phase material suitable for immunoassay. For example, the solid phase may include magnetic particles, paramagnetic particles, membranes, latex particles, tubes, microplates, nylon, polystyrene beads, or combinations thereof.

[0053] In any embodiment, the divalent salt may be produced by a divalent metal cation (such as, but not limited to, Ca). 2+ Mg 2+ Zn 2+ Fe 2+ (etc.) and anions (such as, but not limited to, SO42-) 2- Cl - CO3 2- OH - (etc.) are formed. Exemplary divalent salts include CaSO4, CaCl2, CaOH, CaCO3, MgSO4, MgCl2, MgOH, MgCO3, ZnCl2, and FeCl2. In several aspects, the divalent salt may be MgSO4.

[0054] In several aspects, the concentration of the divalent salt in the solid-phase reagent composition may be greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, or less than or equal to about 60 mM, or from greater than or equal to about 10 mM to less than or equal to about 90 mM, greater than or equal to about 20 mM to less than or equal to about 80 mM, greater than or equal to about 30 mM to less than or equal to about 70 mM, or greater than or equal to about 40 mM to less than or equal to about 60 mM.

[0055] In any embodiment, the solid-phase reagent composition may further comprise a variety of other suitable additional components known in the art and commercially available. Suitable additional components include, but are not limited to, one or more of the following: excipients, sugar alcohols, buffers, antibiotics, blocking agents, or surfactants. “Blocking agent” should be understood as a material capable of blocking specific binding to an interfering agent or non-specific binding to particle surfaces, reaction vessel walls, etc., such as, but not limited to, blocking antibodies, heparin, sorbitol, or bovine serum albumin (BSA). Exemplary additional components include sodium 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonate (sodium DIPSO), tetrasodium ethylenediaminetetraacetic acid (EDTA), heparin, monoclonal blocking antibodies (such as antibodies capable of blocking human anti-mouse antibody (HAMA), antibody mixtures (such as heterophilic blocking reagents (HBR) available from Scantibodies Laboratory, Inc.), and polymeric antibodies (such as PolyMAK-33 available from Roche Diagnostics, and TRU Block available from Meridian Bioscience). TMGentamicin, amphotericin B, normal mouse serum, bovine gamma globulin (BGG), BSA, thiol-modified BSA, sodium azide, sodium cholate, aqueous silicone emulsions (such as Antifoam B emulsion available from Sigma-Aldrich), tert-octylphenoxypolyethoxyethanol (such as Triton available from Sigma-Aldrich) TM X-100), alcohol ethoxylated surfactants (such as ECOSURF available from Dow Chemical) TM EHO-9) and sorbitol (such as D-sorbitol). For example, additional components present in the solid-phase reagent composition may include tetrasodium ethylenediaminetetraacetic acid (EDTA), sorbitol, heparin, and monoclonal blocking antibodies.

[0056] In any embodiment, a solid-phase reagent composition is provided comprising: a solid phase (such as paramagnetic particles) having an anti-CA 19-9 monoclonal antibody or a fragment thereof (such as clone 1116NS19-9 from Fujirebio Diagnostics Inc.) directly or indirectly attached thereto; sodium salt of -[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonate; tetrasodium salt of EDTA; normal mouse serum; thiol-modified BSA; BGG; alcohol ethoxylate surfactant; sorbitol; MgSO4; heparin; monoclonal blocking antibody; sodium azide; gentamicin; and amphotericin B.

[0057] In a further aspect, this document provides liquid chromatography reagent compositions for an immunoassay to detect cancer antigen 19-9 (CA 19-9). The liquid chromatography reagent compositions may comprise conjugates and optionally divalent salts as described herein.

[0058] Examples of conjugates suitable for use in liquid phase reagents include those described in International Patent Publication WO 2022 / 126055, which is incorporated herein by reference in its entirety. For example, the conjugate may comprise an anti-CA 19-9 monoclonal antibody or a fragment thereof as described above, having at least one disulfide bond that can be reduced to two thiol groups and at least one label directly or indirectly attached to said two thiol groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate. In any embodiment, the label is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I: Formula I.

[0059] The conjugation can be referred to as “CA19-9 MAb-PD-HEGAE”, “HEGAE-DiSFPD-Mab-19-9”, or “CA 19-9-MAb-DiSFPD-HEG-DMAE”.

[0060] Optional markers may include the following: Dibromopyridazine dione-dimethylphenyl acridine ester (DiBrPD-DMAE) having the structure of Formula II: Formula II; Dibromopyridazine dione-zwitterion ester (DiBrPD-ZAE) with the structure of Formula III: Formula III; Dibromopyridazine dione-triethylene glycol-trisulfonylpropyl acridine ester (DiBrPD-TEG-TSPAE) with the structure of formula IV: Formula IV; or Dibromopyridazine-2-isopropylacridinium ester with the structure of formula V: Formula V.

[0061] In any embodiment, if present in the liquid phase reagent composition, the divalent salt is a divalent salt as described above. For example, the divalent salt may be MgSO4. The concentration of the divalent salt in the liquid phase reagent composition may be greater than or equal to about 0.5 mM, greater than or equal to about 1 mM, greater than or equal to about 2.5 mM, greater than or equal to about 5 mM, less than or equal to about 15 mM, less than or equal to about 12.5 mM, less than or equal to about 10 mM, or less than or equal to about 7.5 mM, or from greater than or equal to about 0.5 mM to less than or equal to about 15 mM, greater than or equal to about 1 mM to less than or equal to about 10 mM, greater than or equal to about 2.5 mM to less than or equal to about 7.5 mM, or greater than or equal to about 2.5 mM to less than or equal to about 5 mM. In alternative embodiments, the liquid phase reagent composition is also contemplated not to contain the divalent salt as described herein.

[0062] Alternatively or additionally, the liquid phase reagent composition may also contain a variety of other suitable additional components known in the art and commercially available. Suitable additional components include those described above for solid phase reagent compositions (such as excipients, sugar alcohols, buffers, antibiotics, blocking agents, or surfactants). Exemplary additional components include sodium 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonate (sodium DIPSO), tetrasodium ethylenediaminetetraacetic acid (EDTA), heparin, monoclonal blocking antibodies (such as antibodies capable of blocking human anti-mouse antibody (HAMA)), gentamicin, amphotericin B, normal mouse serum, bovine gamma globulin (BGG), bovine serum albumin (BSA), thiol-modified BSA, sodium azide, sodium cholate, aqueous silicone emulsions (such as Antifoam B emulsion available from Sigma-Aldrich), tert-octylphenoxypolyethoxyethanol (such as Triton available from Sigma-Aldrich). TM X-100), alcohol ethoxylated surfactants (such as ECOSURF available from Dow) TM EHO-9), and sorbitol (such as D-sorbitol). For example, additional components present in the liquid phase reagent composition may include tetrasodium ethylenediaminetetraacetic acid (EDTA) and sorbitol.

[0063] In any embodiment, a liquid-phase reagent composition is provided comprising: a conjugate comprising an anti-CA 19-9 monoclonal antibody or a fragment thereof (such as clone 1116NS19-9 from Fujirebio Diagnostics Inc.) having at least one disulfide bond that can be reduced to two thiol groups, and at least one label (such as DiBrPD-HEGAE) directly or indirectly attached to the two thiol groups of the anti-CA 19-9 monoclonal antibody or the fragment thereof; sodium salt of -[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonate; tetrasodium salt of EDTA; thiol-modified BSA; an alcohol ethoxylate surfactant; sorbitol; MgSO4; sodium azide; gentamicin; and amphotericin B.

[0064] C. Immunoassay kit This document also provides an immunoassay kit for detecting cancer antigen 19-9 (CA 19-9). The immunoassay kit comprises a solid-phase reagent composition as described herein and a liquid-phase reagent composition as described herein. For example, the solid-phase reagent composition may comprise a solid-phase carrier as described herein, having an anti-CA 19-9 monoclonal antibody or fragment thereof as described herein directly or indirectly attached thereto, and a divalent salt (such as MgSO4) at a concentration as described herein (e.g., greater than or equal to about 40 mM to less than or equal to about 60 mM). The liquid-phase reagent may comprise a conjugate as described herein, comprising an anti-CA 19-9 monoclonal antibody or fragment thereof as described herein having at least one disulfide bond that can be reduced to two thiol groups, and at least one label (such as DiBrPD-HEGAE) directly or indirectly attached to said two thiol groups of the anti-CA 19-9 monoclonal antibody or fragment thereof. For example, the conjugate may be CA19-9 MAb-PD-HEGAE. In any embodiment, the liquid-phase reagent composition may not contain a divalent salt as described herein. Alternatively, the liquid phase reagent composition may also contain a divalent salt (such as MgSO4) at a concentration as described herein (e.g., greater than or equal to about 1 mM to less than or equal to about 10 mM).

[0065] This document envisions a liquid phase reagent comprising a conjugate as described herein (containing a first analyte-specific binder having at least one labeling agent attached thereto), and a solid phase reagent comprising a solid phase having a second analyte-specific binder directly or indirectly attached thereto. The first and second analyte-specific binders of both the liquid and solid phase reagents can each specifically bind to an epitope of a target analyte to be detected. In a non-limiting embodiment, the epitopes of the target analyte bound by the first and second analyte-specific binders do not substantially overlap, such that the first and second analyte-specific binders can simultaneously bind the same target analyte molecule.

[0066] The assay reagent compositions present in the kit may be provided in any form that allows them to function according to this disclosure. For example, but not limited to, each reagent may be provided in liquid form and presented in the kit in bulk and / or as single aliquots. Alternatively, in certain (but not limiting) embodiments, one or more reagents may be presented in the kit as single aliquots of lyophilized reagents. The use of dried reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0067] In addition to the assay reagents described in detail above, the kit may further contain other reagents for any specific assay described herein or otherwise conceived. The properties of these additional reagents will depend on the specific assay, and their identification is entirely within the skill of a person skilled in the art; therefore, further description is deemed unnecessary. Furthermore, depending on the cross-reactivity and stability of the components / reagents, the components / reagents present in the kit may be individually in separate containers / compartments, or various components / reagents may be combined in one or more containers / compartments. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0068] The kit may contain sufficient reagents for single-use or multiple-use assays. The relative amounts of the various components / reagents in the kit can vary widely to provide concentrations that substantially optimize the reactions required during the assay and further substantially optimize the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit may be provided as dry powders, such as lyophilized powders, and the kit may further include excipients for dissolving the dried reagents; in this way, a reagent solution of appropriate concentration can be obtained from these components for performing the methods or assays according to this disclosure. Non-limiting examples of other reagents that may be included in the kit include washing solutions, diluent solutions, excipients, interfering solutions, positive controls, negative controls, calibrating reagents, mass control reagents, etc. Furthermore, the kit may further include a set of written instructions explaining how to use the kit. Kits of this nature can be used in any method described herein or otherwise contemplated.

[0069] D. Methods for detecting CA 19-9 Methods for detecting cancer antigen 19-9 (CA 19-9) in a sample using solid-phase reagent compositions and liquid-phase reagent compositions as described herein are also provided. Typically, in this method, the sample is simultaneously, wholly, or partially sequentially, with one or more combinations of any conjugates disclosed herein or otherwise contemplated to form a mixture, and the mixture is incubated under conditions that allow the conjugates to bind to CA 19-9 present in the sample, thereby forming a complex; the complex is then detected.

[0070] For example, a sample may be combined simultaneously, wholly, or partially sequentially with the solid-phase reagent composition and liquid-phase reagent composition of the immunoassay kit detailed above to form a mixture. The mixture is then incubated under conditions that allow the solid-phase and liquid-phase reagent compositions to bind to CA 19-9 present in the sample, thereby forming a complex. That is, the analyte-specific binders of the solid and liquid-phase reagent compositions each specifically bind to the epitopes of CA 19-9, and the epitopes are substantially non-overlapping, allowing both analyte-specific binders to bind the same CA 19-9 molecule. The solid-phase reagent-CA 19-9-liquid complex is then detected by any method known in the art.

[0071] Any sample requiring determination of the presence of CA 19-9 can be used as a sample according to the method of this disclosure. Non-limiting examples of samples include biological samples, such as, but not limited to, whole blood or any portion thereof (i.e., plasma (such as EDTA plasma, lithium heparin plasma, etc.) or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder irrigation fluid, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. In exemplary embodiments, the sample may be plasma (such as EDTA plasma) or serum.

[0072] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition can vary; those skilled in the art can determine the specific desired order in which the different components are added to the assay. The simplest order of addition is to add all materials simultaneously and determine the resulting signal. Alternatively, each component or group can be combined sequentially. In some embodiments, an incubation step may be included after one or more additions.

[0073] In any embodiment, the combination and incubation in this method include: a) a first combination step, which includes combining the sample with a solid-phase reagent composition to generate a mixture containing a solid phase, and incubating the mixture containing the solid phase for an appropriate time; and b) a second combination step, which includes combining the mixture containing the solid phase with a liquid-phase reagent composition to generate a mixture, and incubating the mixture for an appropriate time.

[0074] Additionally or optionally, the method may further include one or more additional steps to improve the accuracy and / or precision of the assay. For example (but not limited to), the method may further include one or more washing steps (e.g., single washing, double washing, triple washing, etc.) to remove unbound (or non-specifically bound) reagents from the reaction prior to the detection of complex formation. For example, the method may include washing the mixture containing the solid phase once, twice, or three times with a suitable washing solution to remove excess unbound antigen, followed by resuspending the solid phase particles in a suitable washing solution (e.g., 250 µL of washing solution). After adding the liquid phase reagent composition to generate the mixture, the method may further include washing the mixture once, twice, or three times with a suitable washing solution after incubating the mixture to remove excess unbound antigen. Thus, the method may further include a first separation step comprising separating the solid phase from the mixture containing the solid phase, and a second separation step comprising separating the solid phase from the mixture.

[0075] The conditions for incubating the mixture can vary widely, as long as a complex is formed under those conditions. Immunoassays based on sandwich assay formats are widely practiced, and the conditions for such assays are well known in the art; therefore, selecting suitable detection conditions is entirely within the capabilities of those skilled in the art. For example, incubation temperatures can be from about 5°C to about 99°C, such as about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 99°C. Incubation time can range from approximately 0.2 seconds to approximately 6 hours or overnight, for example, approximately 5 minutes, approximately 10 minutes, approximately 12 minutes, approximately 15 minutes, approximately 30 minutes, approximately 45 minutes, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, or approximately 6 hours. The specific incubation time and temperature may vary depending on the reagents used.

[0076] The specific detection methods employed can vary widely, as long as such methods can detect the complex. Detection of the formed complex using a sandwich assay is widely practiced, and the detection procedures are well known in the art; therefore, selecting a suitable detection method is entirely within the capabilities of a person skilled in the art, and thus further description thereof is considered unnecessary. For example, the methods described herein can be implemented on a Siemens Atellica® IM analyzer or a Siemens ADVIA® Centaur XP system.

[0077] The methods described herein can be implemented in a computer system with a processor that executes specific instructions in a computer program. In some non-limiting embodiments, the computer system may be configured to output the level or amount of CA19-9 in a biological sample. Specifically, the computer program may contain instructions that cause the system to select appropriate subsequent steps, including additional medication, treatment, and / or additional testing for the subject.

[0078] In some non-limiting embodiments, the computer program may be configured to enable the computer system to identify subjects requiring further testing (e.g., additional testing for pancreatic or colorectal cancer), subjects at risk of developing or already diagnosed with pancreatic or colorectal cancer, and / or subjects requiring drug treatment, based on received data (e.g., CA 19-9 levels).

[0079] Figure 5 This is a block diagram of a computer system 1100 that can be used for the operations described above, according to a non-limiting embodiment. System 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. The components 1110, 1120, 1130, and 1140 are interconnected via a system bus 1150. The system may optionally further include an analytical device 1160 for determining the amount of CA 19-9 in a sample.

[0080] In some non-limiting embodiments, processor 1110 is capable of processing instructions executed within system 1100. In one non-limiting embodiment, processor 1110 is a single-threaded processor. In another non-limiting embodiment, processor 1110 is a multi-threaded processor. Processor 1110 is capable of processing instructions stored in memory 1120 or storage device 1130, including receiving or sending information via input / output device 1140.

[0081] In some non-limiting embodiments, memory 1120 stores information within system 1100. In one non-limiting embodiment, memory 1120 is a computer-readable medium. In one non-limiting embodiment, memory 1120 is a volatile memory cell. In another embodiment, memory 1120 is a non-volatile memory cell.

[0082] Storage device 1130 is capable of providing large-capacity storage for system 1100. In one non-limiting embodiment, storage device 1130 is a computer-readable medium.

[0083] Input / output device 1140 provides input / output operations for system 1100. In one non-limiting embodiment, input / output device 1140 includes a keyboard and / or a pointing device. In one non-limiting embodiment, input / output device 1140 includes a display unit for displaying a graphical user interface.

[0084] System 1100 can be used to build a database. In some non-limiting embodiments, the methods of this disclosure are performed in system 1100 located within a chemical analyzer. For example, a computer program product may contain instructions that cause processor 1110 to perform the steps of any method disclosed herein or otherwise contemplated.

[0085] Furthermore, this document also provides a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the methods described herein. For example, a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including any methods disclosed herein or otherwise contemplated. In some non-limiting embodiments, the non-transitory computer-readable medium includes hard disk drives, external hard disk drives, optical disks, CDs, DVDs, and / or the like for storing data. In some non-limiting embodiments, software contained within a physical medium is suitable for the purposes described herein.

[0086] In some non-limiting embodiments, a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including a method for determining the presence of CA 19-9 in a sample, wherein the method includes the steps of: incubating the assay component of this disclosure together with a biological sample and determining the amount of CA 19-9 in the sample. Example

[0087] Examples are provided below. However, it should be understood that the application of this disclosure is not limited to the specific experiments, results, and experimental procedures disclosed herein. Rather, the embodiments are provided merely as one of many implementations and are intended to be exemplary rather than exhaustive.

[0088] An immunoassay for detecting the cancer biomarker CA 19-9 using an antibody-acrididane ester conjugate has previously been developed for the Siemens ADVIA® Centaur XP system; however, this assay is only applicable to serum samples. Therefore, this embodiment relates to the design and manufacture of a novel assay reagent that provides more robust assay performance for samples other than serum (e.g., plasma samples) and can be used for assays performed on the Siemens Atellica® IM analyzer or the Siemens ADVIA® Centaur XP system.

[0089] Example 1: ADVIA Centaur CA 19-9 determination Reagent design and synthesis : In Example 1, the conjugate CA19-9MAb-PD-HEGAE was synthesized according to the description in International Patent Publication WO 2022 / 126055. All reagents were evaluated using a sandwich assay format on a Siemens ADVIA® Centaur XP system.

[0090] The CA 19-9 immunoassay reagent in Example 1 includes a solid-phase (SP) reagent and a liquid-phase reagent (also known as a "luminescent reagent"). Figure 1 The solid-phase reagent contains paramagnetic particles covalently labeled with a monoclonal anti-CA 19-9 antibody (1116-NS-19-9). The luminescent reagent contains a CA19-9 MAb-PD-HEGAE conjugate. The solid and liquid phase reagents form a sandwich assay. The synthesis of the Mab-coated solid phase is a well-known and straightforward process, and therefore its further description is considered unnecessary. Thus, this Example 1 focuses on the creation of novel solid and liquid phase reagents and their performance in CA 19-9 immunoassays. In the currently commercially available ADVIA Centaur CA 19-9 assay, it was found that the CA 19-9 recovered from plasma samples (EDTA plasma) taken from the same patient was higher than that from serum samples. Therefore, in this Example 1, novel solid and liquid phase reagents were designed to mitigate the bias of EDTA plasma. Unexpectedly, it was found that adding an increased amount of MgSO4 to the solid-phase reagent composition resulted in a concentration-specific reduction in the bias of EDTA plasma relative to serum samples. The reagents are prepared as follows.

[0091] Prepare solid-phase diluents using the compositions shown in Table 1 below.

[0092] Table 1: Solid diluents

[0093] Buffer compositions 1-5 containing different concentrations of MgSO4 were prepared using the solid-phase diluents in Table 1, as shown in Table 2 below. Different amounts of MgSO4 were added to ~45 ml of the solid-phase diluents in Table 1, and the volume was subsequently increased to 50 ml using additional solid-phase diluents.

[0094] Table 2: CA 19-9 buffer composition CA 19-9 SP buffer Concentration MgSO4(mM) [Amount of MgSO4 actually used (g)] 1 0 n / a 2 5 0.0306 3 20 0.1257 4 40 0.2417 5 60 0.3662

[0095] Prepare liquid phase reagents using the compositions shown in Table 3 below.

[0096] Table 3: Liquid Phase Reagents

[0097] Paramagnetic particles covalently labeled with monoclonal anti-CA 19-9 antibody were added to 50 mL of CA19-9 SP buffer compositions 1-5 from Table 2 to form individual solid-phase reagents 1-5 with different concentrations of MgSO4.

[0098] Assay description : In one non-limiting embodiment, the ADVIA Centaur CA 19-9 assay is a two-step sandwich immunoassay using direct chemiluminescence technology, employing a single monoclonal antibody 1116-NS-19-9 (from Fujirebio Diagnostics, Inc.) for both the solid-phase and liquid-phase reagents. The antibody is covalently coupled to paramagnetic particles for the solid-phase reagent, while the same clone of antibody is labeled with acridinium ester in the liquid-phase reagent. The system dispenses 75 µL of sample into cuvettes, followed by the addition of 350 µL of solid-phase reagent after 4.75 min. This mixture is incubated at 37°C for 8.25 min, followed by a washing step using ADVIA Centaur Wash Buffer 1 (phosphate-buffered saline with Tween 20 and sodium azide) to remove excess unbound antigen. After the first wash, 100 µL of ADVIA Centaur Wash Buffer 1 is added to resuspend the particles. Subsequently, 100 µL of liquid reagent was reacted with the solid-bound CA 19-9 antigen and incubated for an additional 18 minutes, followed by washing with ADVIA Centaur Wash 1. Then, 300 µL each of acidic reagent (0.5% hydrogen peroxide and 0.45% nitric acid) and alkaline reagent (0.25 M NaOH, 0.45% w / v ARQUAD 16-50) were added to the cuvettes to initiate the chemiluminescent reaction. The concentration of CA 19-9 present in the patient samples was positively correlated with the amount of relative light units (RLU) detected by the system.

[0099] The measurement procedures are summarized in Table 4 below.

[0100] Table 4: Determination Methods

[0101] Results : Using each of the liquid phase reagents and solid phase reagents 1-5 in Table 4, the CA 19-9 immunoassay as described above was performed on three biological samples from the same patient: serum “SST”, lithium heparin plasma (“LiHep”), and EDTA plasma (“EDTA”). The results are shown in Table 5 below.

[0102] Table 5: Recovery of CA 19-9

[0103] As shown in Table 5, when an increased amount of MgSO4 was added to the solid-phase reagent composition, the concentration specificity of the deviation of EDTA plasma from serum was found to decrease.

[0104] Example 2: Atellaca IM CA 19-9 II determination Reagent design and synthesis : In this Example 2, the conjugate HEGAE-DiSFPD-Mab-19-9 was synthesized according to the description in International Patent Publication WO 2022 / 126055. All reagents were analyzed on a Siemens Atellica® IM analyzer using the following methods: Figure 2 Evaluation of the sandwich measurement format illustrated in the figure.

[0105] The CA 19-9 immunoassay reagent in Example 2 includes a solid-phase (SP) reagent and a liquid-phase reagent (also known as a "luminescent reagent"). Figure 2 The solid-phase reagent contains paramagnetic particles covalently labeled with monoclonal anti-CA 19-9 antibody (1116-NS-19-9). The liquid-phase reagent contains a HEGAE-DiSFPD-Mab-19-9 conjugate. The solid and liquid phase reagents form a sandwich assay. The synthesis of the Mab-coated solid phase is a well-known and straightforward process, and therefore its further description is considered unnecessary. Thus, this Example 2 focuses on the creation of novel solid and liquid phase reagents and their performance in CA 19-9 immunoassays. Therefore, in this Example 2, novel solid and liquid phase reagents were designed to mitigate the bias of EDTA plasma. Unexpectedly, it was found that when the solid phase reagent composition includes MgSO4, the bias of EDTA plasma relative to serum samples can be mitigated. The reagents are prepared as follows.

[0106] Solid-phase reagent compositions were prepared using the compositions shown in Table 6 below. Table 6: Solid Phase Reagent Compositions

[0107] In Table 6, “PMP-MAb 19-9” refers to paramagnetic particles covalently labeled with monoclonal anti-CA 19-9 antibody.

[0108] Liquid reagents were prepared using the compositions shown in Table 7 below.

[0109] Table 7: Liquid Phase Reagents

[0110] Assay description : In one non-limiting embodiment, the Atellaca IM CA 19-9 II assay is a two-step sandwich immunoassay using direct chemiluminescence technology, employing a single monoclonal antibody, 1116-NS-19-9 (from Fujirebio Diagnostics, Inc.), for both the solid-phase and liquid-phase reagents. The antibody is covalently coupled to paramagnetic particles for the solid-phase reagent, while an antibody of the same clone is labeled with acridinium ester in the liquid-phase reagent. The system dispenses 75 µL of sample into cuvettes, followed by the addition of 350 µL of the solid-phase reagent. This mixture is incubated at 37°C for 12 min, followed by three independent washes using Atellaca IM washing buffer (phosphate-buffered saline with Tween 20 and sodium azide) to remove excess unbound antigen. After the third wash, 250 µL of water is added to resuspend the particles. Subsequently, 100 µL of liquid reagent was reacted with the solid-bound CA 19-9 antigen and incubated for an additional 12 minutes, followed by three independent washes with Atellaca IM wash buffer. Then, 300 µL each of acidic reagent (0.5% hydrogen peroxide and 0.45% nitric acid) and alkaline reagent (0.25 M NaOH, 0.45% w / v ARQUAD 16-50) were added to cuvettes to initiate the chemiluminescent reaction. The concentration of CA19-9 present in the patient samples was positively correlated with the amount of relative light units (RLU) detected by the system.

[0111] The measurement procedures are summarized in Table 8 below.

[0112] Table 8: Determination Methods

[0113] Results : The CA 19-9 immunoassay was performed using the solid-phase reagents listed in Table 6 and the liquid-phase reagents listed in Table 7, as described above. The results are shown in Tables 9 and 10 below. Figure 3 and Figure 4 Each sample was a matched serum and EDTA plasma sample taken from the same patient. "K2 EDTA plasma" in Table 9 and "K3 EDTA plasma" in Table 10 refer to the potassium (K) present in each EDTA plasma sample tube used for the sample. "K2" refers to dipotassium, and the dipotassium (K2) EDTA plasma tube has two potassium ions chelated with each EDTA molecule. "K3" refers to tripotassium, and the tripotassium (K3) EDTA plasma tube has three potassium ions chelated with each EDTA molecule. Functionally, K2 EDTA or K3 EDTA works by binding calcium ions (Ca). 2+It acts as an anticoagulant. K3 EDTA tubes may also contain preservatives (e.g., small amounts of potassium oxalate or potassium fluoride). The target concentration for K2 EDTA tubes is 1.8 mg K2 EDTA / mL blood, while the target concentration for K3 EDTA tubes is 1.7 mg K3 EDTA / mL blood. For example, a 10 mL K2 EDTA tube contains 18 mg K2 EDTA. Based on the molecular weight ratio of the individual components, this means that the target concentration for K2 EDTA tubes is approximately 0.35 mg potassium / mL blood, and the target concentration for K3 EDTA tubes is approximately 0.45 mg potassium / mL blood.

[0114] Table 9: Recovery of CA 19-9

[0115] Table 10: Recovery of CA 19-9

[0116] Tables 9 and 10 provide CA 19-9 values ​​for measurements of matched serum and plasma (K2 EDTA in Table 9, K3 EDTA in Table 10), showing a very low mean bias between serum and plasma recoveries. As shown in Tables 9 and 10, the bias of EDTA plasma relative to serum is mitigated when a solid-phase reagent composition containing magnesium sulfate (MgSO4) is applied.

[0117] Non-limiting illustrative implementation scheme Illustrative Embodiment 1: A solid-phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9), comprising: a solid phase having an anti-CA 19-9 monoclonal antibody or a fragment thereof directly or indirectly attached thereto; and a divalent salt.

[0118] Illustrative Embodiment 2: The solid-phase reagent composition according to Illustrative Embodiment 1, wherein the concentration of the divalent salt is greater than or equal to about 40 mM to less than or equal to about 60 mM.

[0119] Illustrative Embodiment 3: A solid-phase reagent composition according to any one of the foregoing illustrative embodiments, wherein the divalent salt is MgSO4.

[0120] Illustrative Embodiment 4: The solid-phase reagent composition according to any one of the foregoing illustrative embodiments further comprises one or more of an excipient, a sugar alcohol, a buffer solution, an antibiotic, or a surfactant.

[0121] Illustrative Embodiment 5: The solid-phase reagent composition according to any one of the foregoing illustrative embodiments further comprises tetrasodium ethylenediaminetetraacetic acid (EDTA), sorbitol, heparin, and a monoclonal blocking antibody.

[0122] Illustrative Embodiment 6: A solid-phase reagent composition according to any one of the foregoing illustrative embodiments, wherein the solid phase comprises magnetic particles, paramagnetic particles, membranes, latex particles, tubes, microplates, polystyrene beads, or nylon.

[0123] Illustrative Embodiment 7: A liquid phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9), comprising: a conjugate comprising: an anti-CA 19-9 monoclonal antibody or a fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups; and at least one label having two thiol groups directly or indirectly attached to the anti-CA 19-9 monoclonal antibody or the fragment thereof, thereby forming the conjugate, wherein the label is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I: Formula I; and Divalent salts.

[0124] Illustrative Embodiment 8: The liquid phase reagent composition according to Illustrative Embodiment 7, wherein the concentration of the divalent salt is greater than or equal to about 1 mM to less than or equal to about 10 mM.

[0125] Illustrative Embodiment 9: The liquid-phase reagent composition according to Illustrative Embodiment 7 or 8, wherein the divalent salt is MgSO4.

[0126] Illustrative Embodiment 10: The liquid phase reagent composition according to any one of Illustrative Embodiments 7-9 further comprises one or more of an excipient, a sugar alcohol, a buffer solution, an antibiotic, or a surfactant.

[0127] Illustrative Embodiment 11: The liquid phase reagent composition according to any one of illustrative embodiments 7 to 10 further comprises tetrasodium ethylenediaminetetraacetic acid (EDTA) and sorbitol.

[0128] Illustrative Embodiment 12: An immunoassay kit for detecting cancer antigen 19-9 (CA 19-9) comprising: a) a solid phase reagent comprising: a solid phase having an anti-CA 19-9 monoclonal antibody or a fragment thereof directly or indirectly attached thereto; and a divalent salt; and b) a liquid phase reagent comprising: a conjugate comprising: an anti-CA 19-9 monoclonal antibody or a fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups; and at least one label having two thiol groups directly or indirectly attached to the anti-CA 19-9 monoclonal antibody or a fragment thereof, thereby forming the conjugate, wherein the label is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I: Formula I.

[0129] Illustrative Embodiment 13: The immunoassay kit according to Illustrative Embodiment 12, wherein the concentration of the divalent salt present in the solid phase reagent is greater than or equal to about 40 mM to less than or equal to about 60 mM.

[0130] Illustrative Embodiment 14: An immunoassay kit according to Illustrative Embodiment 12 or 13, wherein the liquid phase reagent further comprises a divalent salt.

[0131] Illustrative Embodiment 15: An immunoassay kit according to any one of illustrative embodiments 12-14, wherein the concentration of the divalent salt present in the liquid phase reagent is greater than or equal to about 1 mM to less than or equal to about 10 mM.

[0132] Illustrative Embodiment 16: An immunoassay kit according to any one of illustrative embodiments 12-15, wherein the divalent salt is MgSO4.

[0133] Illustrative Embodiment 17: An immunoassay kit according to any one of illustrative embodiments 12-16, wherein the immunoassay kit further comprises one or more of a washing solution, a diluent, an excipient, an interference solution, a positive control, a negative control, a calibrator, and a quality control reagent.

[0134] Illustrative Embodiment 18: An immunoassay kit according to any one of illustrative embodiments 12-17, wherein the solid-phase reagent composition and the liquid-phase reagent composition each further comprise one or more excipients, sugar alcohols, buffers, antibiotics, or surfactants.

[0135] Illustrative Embodiment 19: An immunoassay kit according to any one of illustrative embodiments 12-18, wherein the solid phase reagent composition further comprises EDTA tetrasodium salt, sorbitol, heparin and a monoclonal blocking antibody, and the liquid phase reagent composition further comprises EDTA tetrasodium salt and sorbitol.

[0136] Illustrative Embodiment 20: An immunoassay kit according to any one of Illustrative Embodiments 12-19, wherein the solid phase comprises magnetic particles, paramagnetic particles, membranes, latex particles, tubes, microplates, polystyrene beads, or nylon.

[0137] Illustrative Embodiment 21: A method for detecting cancer antigen 19-9 (CA 19-9) in a sample, the method comprising: combining the sample with a solid-phase reagent composition and a liquid-phase reagent composition of the immunoassay kit of claim 12 to form a mixture; incubating the mixture under conditions that allow the solid-phase reagent composition and the liquid-phase reagent composition to bind to CA 19-9 present in the sample, thereby forming a complex; and detecting the complex.

[0138] Illustrative Embodiment 22: The method according to Illustrative Embodiment 21, wherein the combination and the incubation comprise: a) a first combination step comprising combining the sample with a solid-phase reagent composition to generate a mixture containing a solid phase, and incubating the mixture containing the solid phase for an appropriate time; and b) a second combination step comprising combining the mixture containing the solid phase with a liquid-phase reagent composition to generate a mixture, and incubating the mixture for an appropriate time.

[0139] Illustrative embodiment 23: The method according to illustrative embodiment 22 further includes a first separation step, which includes separating a solid phase from the mixture containing the solid phase, and a second separation step, which includes separating a solid phase from the mixture.

[0140] Illustrative Embodiment 24: The method according to any one of Illustrative Embodiments 21-23, wherein the sample comprises plasma or serum.

[0141] Illustrative Embodiment 25: The method according to any one of Illustrative Embodiments 21-24, wherein the sample comprises plasma.

[0142] Illustrative Implementation Scheme 26: The method according to any one of Illustrative Implementation Schemes 21-25, wherein at least one step is performed on an automated analyzer.

[0143] Illustrative embodiment 27: A non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the methods described in any one of illustrative embodiments 21-26.

[0144] Illustrative embodiment 28: A non-transitory computer-readable medium according to illustrative embodiment 27, wherein the instructions cause the processor to control an automated analyzer to perform at least one step of the method.

[0145] Although this disclosure has been described in conjunction with the specific figures, experiments, results, and statements described above, it will be apparent to those skilled in the art that numerous alternatives, modifications, and variations will be apparent. Therefore, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A solid-phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9), comprising: A solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof directly or indirectly attached thereto; and Divalent salts.

2. The solid-phase reagent composition according to claim 1, wherein the concentration of the divalent salt is greater than or equal to about 40 mM to less than or equal to about 60 mM.

3. The solid-phase reagent composition according to claim 1 or claim 2, wherein the divalent salt is MgSO4.

4. The solid-phase reagent composition according to any one of the preceding claims further comprises one or more excipients, sugar alcohols, buffer solutions, antibiotics, or surfactants.

5. The solid-phase reagent composition according to any one of the preceding claims further comprises tetrasodium ethylenediaminetetraacetic acid (EDTA), sorbitol, heparin, and a monoclonal blocking antibody.

6. The solid-phase reagent composition according to any one of the preceding claims, wherein the solid phase comprises magnetic particles, paramagnetic particles, membranes, latex particles, tubes, microplates, polystyrene beads, or nylon.

7. A liquid phase reagent composition for an immunoassay to detect cancer antigen 19-9 (CA 19-9), comprising: Conjugates, comprising: A monoclonal antibody against CA 19-9 or a fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups; and At least one labeling agent directly or indirectly attached to two thiol groups of an anti-CA 19-9 monoclonal antibody or a fragment thereof, thereby forming the conjugate, wherein the labeling agent is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I: Formula I; and Divalent salts.

8. The liquid-phase reagent composition according to claim 7, wherein the concentration of the divalent salt is greater than or equal to about 1 mM to less than or equal to about 10 mM.

9. The liquid-phase reagent composition according to claim 7 or claim 8, wherein the divalent salt is MgSO4.

10. The liquid phase reagent composition according to any one of claims 7-9, further comprising one or more excipients, sugar alcohols, buffer solutions, antibiotics, or surfactants.

11. The liquid phase reagent composition according to any one of claims 7-10, further comprising tetrasodium ethylenediaminetetraacetic acid (EDTA) and sorbitol.

12. An immunoassay kit for detecting cancer antigen 19-9 (CA 19-9), comprising: a) A solid-phase reagent, comprising: A solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof directly or indirectly attached thereto; and Divalent salts; and b) Liquid phase reagents, comprising: Conjugates, comprising: A monoclonal antibody or fragment thereof having at least one disulfide bond capable of being reduced to two thiol groups; and At least one labeling agent directly or indirectly attached to two thiol groups of an anti-CA 19-9 monoclonal antibody or a fragment thereof, thereby forming the conjugate, wherein the labeling agent is dibromopyridazine dione-hexa(ethylene) glycol-dimethylphenyl acridine ester (DiBrPD-HEGAE) having the structure of Formula I: Formula I.

13. The immunoassay kit of claim 12, wherein the concentration of the divalent salt present in the solid-phase reagent is greater than or equal to about 40 mM to less than or equal to about 60 mM.

14. The immunoassay kit according to claim 13, wherein the liquid phase reagent further comprises a divalent salt.

15. The immunoassay kit of claim 14, wherein the concentration of the divalent salt present in the liquid phase reagent is greater than or equal to about 1 mM to less than or equal to about 10 mM.

16. The immunoassay kit according to any one of claims 12-15, wherein the divalent salt is MgSO4.

17. The immunoassay kit according to any one of claims 12-16, wherein the immunoassay kit further comprises one or more of washing solution, dilution solution, excipient, interference solution, positive control, negative control, calibration reagent and quality control reagent.

18. The immunoassay kit according to any one of claims 12-17, wherein the solid-phase reagent composition and the liquid-phase reagent composition each further comprise one or more excipients, sugar alcohols, buffer solutions, antibiotics, or surfactants.

19. The immunoassay kit according to any one of claims 12-18, wherein the solid phase reagent composition further comprises EDTA tetrasodium salt, sorbitol, heparin and a monoclonal blocking antibody, and the liquid phase reagent composition further comprises EDTA tetrasodium salt and sorbitol.

20. The immunoassay kit according to any one of claims 12-19, wherein the solid phase comprises magnetic particles, paramagnetic particles, membranes, latex particles, tubes, microplates, polystyrene beads, or nylon.

21. A method for detecting cancer antigen 19-9 (CA 19-9) in a sample, the method comprising: The sample is combined with the solid-phase reagent composition and liquid-phase reagent composition of the immunoassay kit of claim 12 to form a mixture; The mixture is incubated under conditions that allow the solid-phase reagent composition and the liquid-phase reagent composition to bind with CA 19-9 present in the sample, thereby forming a complex; as well as The complex was detected.

22. The method of claim 21, wherein the combination and the incubation comprise: a) A first combination step, which includes combining the sample with a solid-phase reagent composition to produce a mixture containing a solid phase, and incubating the mixture containing the solid phase for an appropriate time; and b) A second combination step, comprising combining the solid-containing mixture with a liquid-phase reagent composition to produce a mixture, and incubating the mixture for an appropriate time.

23. The method of claim 22, further comprising a first separation step comprising separating a solid phase from the mixture containing the solid phase, and a second separation step comprising separating a solid phase from the mixture.

24. The method according to any one of claims 21-23, wherein the sample comprises plasma or serum.

25. The method according to any one of claims 21-24, wherein the sample comprises plasma.

26. The method according to any one of claims 21-25, wherein at least one step is performed on an automated analyzer.

27. A non-transitory computer-readable medium comprising executable instructions that, when executed, cause a processor to perform operations including the method of any one of claims 21-26.

28. The non-transitory computer-readable medium of claim 27, wherein the instructions cause the processor to control the automated analyzer to perform at least one step of the method.

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