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

Abstract

Reagents for immunoassays for detecting cancer antigen 19-9 (CA 19-9) are provided herein, which include a divalent salt, such as MgSO4. Also provided are kits including the reagents as well as methods and systems for detecting CA 19-9 in a sample, such as a plasma or a serum sample.

Description

REAGENT COMPOSITIONS FOR IMMUNOASSAYS AND USE OF THE SAME IN DETECTION OF CANCER ANTIGEN 19-9 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 595,388 filed on 2 November 2023. The entire disclosure of the above application is incorporated herein by reference. 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 a biological sample. In particular, the disclosure relates to reagents, such as solid phase reagent compositions including a divalent salt (e.g., MgSO4) and a solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof directly or indirectly attached thereto and use of such reagents for detecting CA 19-9. BACKGROUND

[0003] This section provides background information related to the present disclosure which is not necessarily prior art.

[0004] Pancreatic cancer is difficult to diagnose early because of a lack of signs or symptoms. By the time symptoms begin to be observed, the tumors have often grown very large or have already spread outside the pancreas. As such, pancreatic cancer has one of the lowest survival rates of all cancers.

[0005] Tumor markers are useful in the diagnosis and management of various cancers. In particular (but not by way of limitation), cancer antigen 19-9 (CA 19-9) is a tumor marker for pancreatic and colorectal carcinoma. CA 19-9 is a glycoprotein, the sialylated form of the Lewis blood group antigen, and exists as a mucin in serum. CA 19-9 is synthesized by normal human pancreatic and biliary ductular cells and by gastric, colon, endometrial, and salivary epithelia, and elevated levels of CA 19-9 in serum can be an indication of pancreatic, gastric, and hepatobiliary malignancies. The original monoclonal antibody against CA 19-9 was developed from a human colon carcinoma cell line, as published in 1979 (Koprowski, et al. (1979) Somatic Cell Genet., 5:957-971), and different immunoassays for CA 19-9 have since been developed and are commercially available. The performance characteristics of these commercially available CA 19- 9 assays have also been assessed (see, for example (but not by way of limitation) 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 the commercially available CA 19-9 assays include the ADVIA®Centaur CA 19-9 assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY); Atellica 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® Dxl 800 Gl Monitor (Beckman Coulter, Fullerton, CA).

[0006] Despite the availability of commercially available immunoassays, the sensitivity and accuracy observed for these assays requires improvement. For example, assays that use multiple forms of biological samples, such as serum, ethylenediaminetetraacetic acid (EDTA) plasma, lithium heparin plasma, etc., should demonstrate substantially equivalent recovery of CA 19-9 across sample types. However, it has been found that the matrix of various biological sample types often varies and can interfere with detection accuracy in the assay.

[0007] Thus, there is a need in the art for new and improved reagent compositions for use in immunoassays that overcome the disadvantages of current assays. It is to such reagent compositions, as well as kits and methods of using the same, that the present disclosure is directed. SUMMARY

[0008] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

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

[0010] In yet other aspects, the present disclosure provides a liquid phase reagent composition for an immunoassay for detecting cancer antigen 19-9 (CA 19-9). The liquid phase reagent composition includes a conjugate and a divalent salt, such as MgSO4. The conjugate includes an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate, wherein the label is a dibromopyridazinedione- hexa(ethylene)glycol-dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I.

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

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

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

[0014] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0016] FIG.1 schematically depicts one non-limiting embodiment of an assay format and principle for an immunoassay for the detection of the cancer antigen CA 19-9 in accordance with the present disclosure in Example 1. The immunoassay employs a solid phase reagent that includes a divalent salt and paramagnetic particles (PMP) covalently labeled with a monoclonal anti-CA 19-9 antibody and a liquid phase reagent that includes an anti-CA 19-9 monoclonal antibody- acridinium ester (CA 19-9- MAb-AE) conjugate.

[0017] FIG.2 schematically depicts one non-limiting embodiment of an assay format and principle for an immunoassay for the detection of the cancer antigen CA 19-9 in accordance with the present disclosure in Example 2. The immunoassay employs a solid phase reagent that includes a divalent salt and paramagnetic particles (PMP) covalently labeled with a monoclonal anti-CA 19-9 antibody and a liquid phase reagent that includes an anti-CA 19-9 monoclonal antibody- acridinium ester (CA 19-9- MAb-AE) conjugate.

[0018] FIG.3 is a plot of CA 19-9 measured values from K2 EDTA plasma vs. CA 19-9 measured values from matched samples from the assay of Example 2.

[0019] FIG.4 is a plot of CA 19-9 measured values from K3 EDTA plasma vs. CA 19-9 measured values from serum results from matched samples from the assay of Example 2.

[0020] FIG. 5 depicts an exemplary block diagram of a computer system suitable for executing the methods of the present disclosure on a chemical analyzer. DETAILED DESCRIPTION

[0021] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. A. Definitions

[0022] The terminology used herein is for the purpose of describing particular example 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 specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0023] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0024] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0025] 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 limitsmay 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.

[0026] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[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] Throughout this disclosure, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0029] 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.

[0030] 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.

[0031] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges.

[0032] The term “conjugate” refers to any substance formed from the joining together of two parts. Representative conjugates in accordance with the present disclosure include those formed by the joining together of a small molecule and a large molecule, such as a protein.

[0033] 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 or serum), urine, saliva, 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, combinations thereof, and the like.

[0034] 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.

[0035] The term "antibody" (abbreviated “Ab”) is used in the broadest sense, and specifically (but not by way of limitation) covers monoclonal antibodies (abbreviated “MAb”) (including full length monoclonal antibodies), polyclonal antibodies (abbreviated “pAb”), multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired biological activity of analyte binding. Thus, the term "antibody" or "antibody peptide(s)" refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen- binding fragments include Fab, Fab', F(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 or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, e.g., Hudson et al. (Nature Med. (2003) 9:129-134). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).

[0039] The term "antigen binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "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.

[0036] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a "conformational epitope"), as well as specific charge characteristics.

[0037] 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 include 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.

[0038] 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 includes 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 include that particular epitope or closely related epitopes. In certain instances, for example, homologous proteins from different species may include 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 receptoror 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).

[0039] An "isolated" antibody is one which has been separated and / or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In certain embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequentator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, alternatively, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the environment in which the antibody is produced will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In addition, the "isolated antibody" is substantially free of other antibodies having different antigenic specificities. An isolated antibody may, however, have some cross-reactivity to other, related antigens.

[0040] 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 including the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that in one method of production they may be synthesized by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, in one embodiment, the monoclonal antibodies produced in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0041] 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.

[0042] 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 includes at least one antigenic determinant or "epitope," which is the region of the analyte which binds to the analyte- specific binding partner (i.e., antibody).

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

[0044] The term “polypeptide” refers to any compound formed by the linkage of two or more amino acids via an amide bond. Representative polypeptides include polymers of α-amino acids in which the α-amino group of each non-terminal amino acid residue is linked to the α- carboxyl group of an adjacent residue in a linear chain. High molecular weight polypeptides are referred to as “proteins.”

[0045] The term “label” refers to a member of a signal producing system. The label is capable of being detected directly or is detectable through a specific binding reaction that produces a detectable signal. For example, a label may be an identifying tag that can be attached to a carrier substance or molecule to detect an analyte. The labels generally are radioisotopic, luminescent, particulate, or enzymic. The label can be a poly(amino acid), or protein, or non-poly(amino acid), isotopic or non-isotopic, usually non-isotopic, and can be a catalyst, such as an enzyme (e.g., β- galactosidase, peroxidase, etc.), a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule (e.g., rhodamine, fluorescein isothiocyanate or FITC, etc.), chemiluminescent molecule(e.g., dioxetanes, luciferin, etc.), coenzyme, enzyme substrate, radioactive group (e.g.,125I), a protein-binding partner (e.g., biotin), a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like.

[0046] The term “non-poly(amino acid) labels” refers to those labels that are not proteins such as enzymes. A non-poly(amino acid) label may be a member of a signal producing system.

[0047] The signal producing system may have one or more components, at least one component being the label. The signal producing system generates a signal that relates to the presence of an entactogen in a sample. The signal producing system includes all of the reagents required to produce a measurable signal. Other components of the signal producing system may be included in a developer solution and can include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, and the like. Other components of the signal producing system may be coenzymes, substances that react with enzymic products, other enzymes and catalysts, and the like. The signal producing system provides a signal detectable by external means, by use of electromagnetic radiation, desirably by visual examination. Exemplary signal-producing systems are described in U.S. Pat. No.5,508,178 (Rose, et al.), the relevant disclosure of which is incorporated herein by reference.

[0048] The phrase “detecting an analyte” refers to any quantitative, semi-quantitative, or qualitative method, as well as to all other methods for determining an analyte in general, and CA 19-9 in particular. The terms “detecting,” “determining,” “identifying,” and the like are used synonymously herein, and all lie within the scope of the present disclosure.

[0049] The phrase “reagent kit” or term “kit” refers to an assembly of materials that are used in performing an assay. The reagents can be provided in packaged combination in the same or in separate containers, depending on their cross-reactivities and stabilities, and in liquid or in lyophilized form. The amounts and proportions of reagents provided in the kit can be selected so as to provide optimum results for a particular application. A reagent kit embodying features of the present disclosure includes antibodies specific for CA 19-9, conjugates thereof, and / or enzymes or proteins necessary for detecting the presence and quantity of CA 19-9 or complexes thereof in a sample. The kit may further include calibration and control materials. The reagents may remain 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 an analyte to be measured. A sample suspected ofcontaining an analyte and the corresponding calibration material are assayed under similar conditions. The concentration of analyte is calculated by comparing the results obtained for the unknown specimen with the results obtained for the standard. This is commonly done by constructing a calibration curve. B. Reagent Compositions for Immunoassays

[0051] Reagent compositions for an immunoassay for detecting cancer antigen 19-9 (CA 19-9) are provided herein. As discussed above, the matrix of various biological sample types often varies and can interfere with detection accuracy in the assay. For example, it has been found that plasma samples (e.g., EDTA plasma) in CA 19-9 assays recovered higher CA 19-9 than serum samples resulting in undesirable EDTA plasma bias. To address this problem, a reagent composition including a divalent salt is provided which advantageously mitigates EDTA plasma bias in plasma samples. Without being bound by theory, it is believed that inclusion of a divalent salt, such as magnesium sulfate (MgSO4), can mitigate EDTA plasma bias as EDTA has a higher binding affinity for divalent cations, such as Mg2+. Thus, reagent compositions, such as solid phase and liquid phase reagent compositions, including a divalent salt are provided herein. It is contemplated herein that the reagent compositions provided are not limited to use in assays with plasma samples but are suitable for use in assays with various biological samples as described herein.

[0052] In any embodiment, a solid phase reagent composition includes a solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof (also referred to as “CA 19-9 MAb” or “MAb 19-9”) directly or indirectly attached thereto and a divalent salt. Anti-CA 19-9 monoclonal antibodies / fragments thereof are known in the art and are commercially available; for example (but not by way of limitation), anti-CA 19-9 monoclonal antibodies that can be utilized in accordance with the present disclosure can be obtained from Abeam (Cambridge, UK); Abnova Corporation (Walnut, CA); Agilent Technologies (Santa Clara, CA); Antibodies- Online Inc. (Limerick, PA); Biocare Medical (Pacheco, CA); Biorbyt Ltd. (St. Louis, MO); Creative Diagnostics (Shirley, NY); Cell Sciences (Newburyport, MA); EastCoast Bio (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); Thermo Fisher Scientific (Waltham, MA); US Biological Life Sciences (Salem, MA); and many others. For example, the anti-CA 19-9 MAb (Catalog No: 210-580; clone 1116NS19-9) from Fujirebio Diagnostics Inc. (Malvern, PA), or any of the other anti-CA 19-9 monoclonal antibodies / fragments disclosed above may be used. Any suitable solid phase materialfor use in an immunoassay may be included in the solid phase reagent. For example, the solid phase may include magnetic particles, paramagnetic particles, a membrane, latex particles, a tube, a microwell plate, nylon, polystyrene bead(s), or a combination thereof.

[0053] In any embodiment, the divalent salt can be formed from a divalent metal cation, such as, but not limited to, Ca2+, Mg2+, Zn2+, Fe2+, and the like, along with an anion, such as, but not limited, to SO42-, Cl-, CO32-, OH-, and the like. Exemplary divalent salts include CaSO4,CaCl2, CaOH, CaCO3, MgSO4, MgCl2, MgOH, MgCO3, ZnCl2, and FeCl2. In various aspects, the divalent salt may be MgSO4.

[0054] In various aspects, the divalent salt may be present in the solid phase reagent composition in a concentration of 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 also include various other suitable additional components known in the art and commercially available. Suitable additional components include, but are not limited to, one or more of: an excipient, a sugar alcohol, a buffer, an antibiotic, a blocker, or a surfactant. A “blocker” is understood to be a material that can block specific binding to interferents or nonspecific binding to the particle surface, reaction vessel walls, etc., for example, materials such as, but not limited to, a blocking antibody, heparin, sorbitol, or bovine serum albumin (BSA). Exemplary additional components include 3-[N,N- bis(hydroxyethyl)amino]-2-hydroxypropanesulphonic acid sodium salt (DIPSO sodium salt), ethylenediaminetetraacetic acid (EDTA) tetrasodium, heparin, a monoclonal blocking antibody, such as an antibody capable of blocking human anti-mouse antibody (HAMA), a mixture of antibodies such as Heterophilic Blocking Reagent (HBR) available from Scantibodies Laboratory, Inc., a polymerized antibody such as PolyMAK-33 available from Roche Diagnostics, TRU BlockTMavailable from Meridian Bioscience, gentamicin, amphotericin B, normal mouse serum, bovine gamma globulin (BGG), BSA, sulfhydryl-modified BSA, sodium azide, sodium cholate, aqueous silicone emulsion (e.g., Antifoam B emulsion available from Sigma-Aldrich), t- octylphenoxypolyethoxyethanol (e.g., TritonTMX-100 available from Sigma-Aldrich), alcohol ethoxylate surfactant (e.g., ECOSURFTMEH0-9 available from Dow Chemical), and sorbitol (e.g.,D-sorbitol). For example, additional components present in the solid phase reagent composition may include ethylenediaminetetraacetic acid (EDTA) tetrasodium, sorbitol, heparin, and a monoclonal blocking antibody.

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

[0057] In further aspects, a liquid phase reagent composition for an immunoassay for detecting cancer antigen 19-9 (CA 19-9) is provided herein. The liquid phase reagent composition may include a conjugate and optionally, a divalent salt as described herein.

[0058] Examples of suitable conjugates for use in the liquid phase reagent include those as described in International Patent Publication WO 2022 / 126055, which is hereby incorporated by reference in its entirety. For example, the conjugate may include an anti-CA 19-9 monoclonal antibody or fragment thereof as described above having at least one disulfide bond capable of reduction to two sulfhydryl groups and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate. In any embodiment, the label is a dibromopyridazinedione-hexa(ethylene)glycol- dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I. The conjugate may be referred to as “CA19-9 MAb-PD-HEGAE” or “HEGAE-DiSFPD-Mab-19- 9” or “CA 19-9-MAb-DiSFPD-HEG-DMAE.”

[0059] Alternative labels may include the following: a dibromopyridazinedione-dimethylphenyl acridinium ester (DiBrPD-DMAE) having the structure of Formula II:a dibromopyridazinedione-zwitterionic acridinium ester (DiBrPD-ZAE) having the structure of Formula III:a dibromopyridazinedione-triethylene glycol- tri-sulfo propyl acridinium ester (DiBrPD- TEG-TSPAE) having the structure of Formula IV:Formula IV; or a dibromopyridazinedione-2-isopropyl acridinium ester (DiBrPD-ISOZAE) having the structure of Formula V:

[0060] In any embodiment, the divalent salt, if present in the liquid phase reagent composition, is a divalent salt as described above. For example, the divalent salt may be MgSO4. The divalent salt may be present in the liquid phase reagent composition in a concentration of 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, it is also contemplated herein that the liquid phase reagent composition does not include a divalent salt as described herein.

[0061] Additionally or alternatively, the liquid phase reagent composition may alsoinclude various other suitable additional components known in the art and commercially available. Suitable additional components include those as described above with respect to the solid phase reagent composition (e.g., an excipient, a sugar alcohol, a buffer, an antibiotic, a blocker, or a surfactant). Exemplary additional components include 3-[N,N-bis(hydroxyethyl)amino]-2- hydroxypropanesulphonic acid sodium salt (DIPSO sodium salt), ethylenediaminetetraacetic acid (EDTA) tetrasodium, heparin, a monoclonal blocking antibody, e.g., antibody capable of blocking human anti-mouse antibody (HAMA), gentamicin, amphotericin B, normal mouse serum, bovine gamma globulin (BGG), bovine serum albumin (BSA), sulfhydryl-modified BSA, sodium azide, sodium cholate, aqueous silicone emulsion (e.g., Antifoam B emulsion available from Sigma- Aldrich), t-octylphenoxypolyethoxyethanol (e.g., TritonTMX-100 available from Sigma-Aldrich), alcohol ethoxylate surfactant (e.g., ECOSURFTMEH0-9 available from Dow), and sorbitol (e.g., D-sorbitol). For example, additional components present in the liquid phase reagent composition may include ethylenediaminetetraacetic acid (EDTA) tetrasodium and sorbitol.

[0062] In any embodiment, a liquid phase reagent composition is provided which includes the following: a conjugate including an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups (e.g., clone 1116NS19-9 from Fujirebio Diagnostics Inc) and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof (e.g., DiBrPD-HEGAE); -[N,N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulphonic acid sodium salt; EDTA tetrasodium, sulfhydryl-modified BSA; alcohol ethoxylate surfactant; sorbitol; MgSO4; sodium azide; gentamicin; and amphotericin B. C. Immunoassay Kits

[0063] Immunoassay kits for detecting cancer antigen 19-9 (CA 19-9) are also provided herein. The immunoassay kit includes 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 include a solid phase 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 (e.g., MgSO4) in 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 include a conjugate as described herein including an anti-CA 19-9 monoclonal antibody or fragment thereof as described herein having at least one disulfide bond capable of reduction to two sulfhydryl groups and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof (e.g., DiBrPD-HEGAE). For example, the conjugate may be CA19-9 MAb-PD-HEGAE. In any embodiment, the liquid phase reagentcomposition may not include a divalent salt as described herein. Alternatively, the liquid phase reagent composition may include a divalent salt (e.g., MgSO4) in a concentration as described herein (e.g., greater than or equal to about 1 mM to less than or equal to about 10 mM).

[0064] It is contemplated herein that the liquid phase reagent includes a conjugate as described herein (containing a first analyte-specific binder having at least one label attached thereto) and the solid phase reagent includes a solid phase having a second analyte-specific binder directly or indirectly attached thereto. Each of the first and second analyte-specific binders of the liquid phase and solid phase reagents may specifically bind to an epitope of the target analyte to be detected. In a non-limiting embodiment, the epitopes of the target analyte to which the first and second analyte-specific binders bind do not substantially overlap, such that the first and second analyte-specific binders can bind simultaneously to the same target analyte molecule.

[0065] The assay reagent compositions present in the kits 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(s) within the kit. Alternatively, in a particular (but non-limiting) embodiment, one or more of the reagents may be disposed in the kit in the form of a single aliquot lyophilized reagent. The use of dried reagents in 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.

[0066] In addition to the assay reagents described in detail above, the kits may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein. 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. Also, the components / reagents present in the kits may each be in separate containers / compartments, or various components / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. In addition, the kit may include a microfluidics device in which the components / reagents are disposed.

[0067] The kit may include reagents sufficient for a single use assay or for a multiple use assay. The relative amounts of the various components / reagents in the kits can vary widely to provide for concentrations of the components / reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity of an assay. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further includeexcipient(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 components. Non-limiting examples of other reagents that can be included in the kits include wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, calibration reagents, quality control reagents, and the like. In addition, the kit can further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein. D. Methods for Detecting CA 19-9

[0068] Methods for detecting cancer antigen 19-9 (CA 19-9) in a sample are also provided, which utilize the solid phase reagent composition as described herein and the liquid phase reagent composition as described herein. Generally, in the method, a sample is combined, either simultaneously or wholly or partially sequentially, with one or more of any of the conjugates disclosed or otherwise contemplated herein to form a mixture, and the mixture is incubated under conditions that allow for binding of the conjugate to CA 19-9 present in the sample, thereby forming a complex; then, the complex is detected.

[0069] For example, a sample may be combined, either simultaneously or wholly or partially sequentially, with the solid phase reagent composition and the liquid phase reagent composition of the immunoassay kit described in detail herein above to form a mixture. The mixture is then incubated under conditions that will allow for binding of the solid phase reagent composition and the liquid phase reagent composition to CA 19-9 present in the sample, thereby forming a complex. That is, each of the analyte-specific binders of the solid phase and the liquid phase reagent compositions specifically binds to an epitope of CA 19-9, and the epitopes do not substantially overlap, such that the two analyte-specific binders can bind to the same CA 19-9 molecule. The complex of solid phase reagent-CA 19-9-liquid phase may then be detected by any method known in the art.

[0070] Any sample for which an assay for the presence of CA 19-9 is desired can be utilized as the sample in accordance with the methods of the present disclosure. Non-limiting examples of samples include a biological sample such as, but not limited to, whole blood or any portion thereof (i.e., plasma (e.g., EDTA plasma, lithium heparin plasma, etc.) or serum), urine, saliva, 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 combinations thereof. In an exemplary embodiment, the sample may be plasma (e.g., EDTA plasma) or serum.

[0071] As mentioned above, the various components of the method are provided in combination (either simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components may be varied; a person having ordinary skill in the art can determine the particular desired order of addition of the different components to the assay. The simplest order of addition is to add all the materials simultaneously and determine the signals produced therefrom. Alternatively, each of the components, or groups of components, can be combined sequentially. In certain embodiments, an incubation step may be involved subsequent to one or more additions.

[0072] In any embodiment, the combining and the incubating in the method includes: a) a first combining step including combining the sample with the solid phase reagent composition to produce a solid phase-containing mixture and incubating the solid phase-containing mixture for a suitable amount of time; and b) a second combining step including combining the solid phase- containing mixture with the liquid phase reagent composition to produce the mixture and incubating the mixture for a suitable amount of time.

[0073] Additionally or alternatively, the method may further include one or more additional steps to increase the accuracy and / or precision of the assay. For example (but not by way of limitation), the method may further include one or more wash steps (e.g., one wash, two washes, three washes, etc.) for removing unbound (or non-specifically bound) reagent from the reaction prior to detection of complex formation. For example, the method may include washing the solid phase-containing mixture one, two, or three times with a suitable wash solution to remove excess unbound antigens followed by resuspension of the solid particles in a suitable wash solution (e.g., 250 µL of wash solution). After addition of the liquid phase reagent composition to produce the mixture, the method may further include, washing the mixture one, two, or three times with a suitable wash solution to remove excess unbound antigens after incubation of the mixture. Thus, the method may further include a first separation step including separating the solid phase from the solid phase-containing mixture and a second separation step including separating the solid phase from the mixture.

[0074] The conditions under which the mixture is incubated can vary widely, so long as the complex is formed under such conditions. Immunoassays based on a sandwich assay format are widely performed and immunoassay conditions are well known in the art; thus, selection of appropriate assay conditions is well within the purview of a person having ordinary skill in the art. For example, incubation temperatures may be 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 times may be about 0.2 sec to about 6 h or overnight, for example about 5 min, about 10 min, about 12 min, about 15 min, about 30 min, about 45 min, about 1 h, about 1.5 h, about 2 h, about 3 h, about 4 h, about 5 h, or about 6 h. The specific time and temperature of the incubation may depend on the reagents used.

[0075] The particular detection method utilized can vary widely, so long as the complex can be detected under such methods. Detection of complexes formed in a sandwich assay format are widely performed, and the detection procedures are well known in the art; thus, selection of appropriate detection methods is well within the purview of a person having ordinary skill in the art, and thus no further description thereof is deemed necessary. For example, the methods described herein may be performed on a Siemens Atellica® IM analyzer or a Siemens ADVIA® Centaur XP system.

[0076] Methods described herein can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some non-limiting embodiments, a computer system may be arranged to output a level or amount of CA 19-9 in a biological sample. Particularly, a computer program may include instructions for the system to select appropriate next steps, including additional medication, a treatment, and / or additional testing for a subject.

[0077] In some non-limiting embodiments, the computer program may be configured such that the computer system can identify a subject for further testing (e.g., additional tests for pancreatic or colorectal carcinoma), identify a subject as being at risk of having or having pancreatic or colorectal cancer, and / or identify a subject to receive medication based on received data (e.g., CA 19-9 amount).

[0078] FIG. 5 is a block diagram of a computer system 1100 that can be used in the operations described above, according to one non-limiting embodiment. The system 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Each of the components 1110, 1120, 1130, and 1140 are interconnected using a system bus 1150. The system may optionally further include analyzing equipment 1160 for determining an amount of CA 19-9 in a sample.

[0079] In certain non-limiting embodiments, the processor 1110 is capable of processing instructions for execution within the system 1100. In one non-limiting embodiment, the processor 1110 is a single-threaded processor. In another non-limiting embodiment, the processor 1110 is a multi-threaded processor. The processor 1110 is capable of processing instructions stored in the memory 1120 or on the storage device 1130, including for receiving or sending information through the input / output device 1140.

[0080] In certain non-limiting embodiments, the memory 1120 stores information within the system 1100. In one non-limiting embodiment, the memory 1120 is a computer-readable medium. In one non-limiting embodiment, the memory 1120 is a volatile memory unit. In another embodiment, the memory 1120 is a non-volatile memory unit.

[0081] The storage device 1130 is capable of providing mass storage for the system 1100. In one non-limiting embodiment, the storage device 1130 is a computer-readable medium.

[0082] The input / output device 1140 provides input / output operations for the system 1100. In one non-limiting embodiment, the input / output device 1140 includes a keyboard and / or pointing device. In one non-limiting embodiment, the input / output device 1140 includes a display unit for displaying graphical user interfaces.

[0083] The system 1100 can be used to build a database. In certain non-limiting embodiments, a method of the present disclosure is performed in the system 1100 disposed within a chemical analyzer. For example, a computer program product can include instructions that cause the processor 1110 to perform the steps of any of the methods disclosed or otherwise contemplated herein.

[0084] Additionally, non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as described herein are provided. For example, a non-transitory computer readable medium containing executable instructions that when executed can cause a processor to perform operations including any of the methods disclosed or otherwise contemplated herein. In certain non-limiting embodiments, a non-transitory computer readable medium includes a hard drive, external hard drive, discs, CDs, DVDs, and / or the like that stores data. In certain non-limiting embodiments, software disposed within a physical medium is suitable for use herein.

[0085] In some non-limiting embodiments, a non-transitory computer readable media is disclosed that contains executable instructions that when executed cause a processor to perform operations including a method of determining the presence of CA 19-9 in a sample, wherein the method includes the steps of: incubating the assay components of the present disclosure with a biological sample, and determining the amount of CA 19-9 in a sample. EXAMPLES

[0086] 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. Rather, the Example is simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0087] An immunoassay for the detection of cancer marker CA 19-9 employing anantibody-acridinium ester conjugate was previously developed for the Siemens ADVIA® Centaur XP system, but the assay is only for use with serum samples. Therefore, the present Examples involved the design and production of new assay reagents that provide more robust assay performance with samples other than serum, for example, plasma samples and which can be used in assays performed on a Siemens Atellica® IM analyzer or a Siemens ADVIA® Centaur XP system. Example 1- ADVIA Centaur CA 19-9 Assay Reagent Design and Syntheses:

[0088] In this Example 1, a conjugate, CA19-9 MAb-PD-HEGAE, was synthesized as described in International Patent Publication WO 2022 / 126055. All reagents were evaluated on the Siemens ADVIA® Centaur XP system using a sandwich assay format.

[0089] The CA 19-9 immunoassay reagents in this Example 1 included one solid phase (SP) reagent and one liquid phase reagent (also referred to as “lite reagent”) (FIG. 1). The solid phase reagent contains paramagnetic particles covalently labeled with monoclonal anti-CA 19-9 antibody (1116-NS-19-9). The lite reagent contains the CA19-9 MAb-PD-HEGAE conjugate. Both solid phase and liquid phase reagents form the sandwich assay. The synthesis of MAb-coated solid phase is a well-known and straight forward process, and therefore no further description thereof is deemed necessary. As such, this Example 1 focuses on the creation of new solid phase and liquid phase reagents and their performance in a CA 19-9 immunoassay. In the current commercial ADVIA Centaur CA 19-9 assay, it was found that a plasma sample (EDTA plasma) recovered higher CA 19-9 than serum samples drawn at the same time from the sample patients. Thus, in the present Example 1, new solid phase reagents and liquid phase reagents were designed which can mitigate EDTA plasma bias. It was surprisingly found that a concentration-specific reduction in EDTA plasma bias relative to serum samples can be achieved when increasing amounts of MgSO4were added to the solid phase reagent composition. The reagents were prepared as follows.

[0090] A solid phase diluent was prepared with the composition shown below in Table 1. Table 1. Solid Phase Diluent Component Amount UnitsComponent Amount Units Amphotericin B

[0091] BgSO4were prepared using the solid phase diluent of Table 1 as shown in Table 2 below. MgSO4 in various amounts was added to ~45 ml of the solid phase diluent of Table 1 and then the volume was increase to 50 ml with additional solid phase diluent. Table 2. CA 19-9 Buffer Compositions Actual CA 19-9 Concentration Amt. 4

[0092] A liquid phase reagent was prepared with the following composition shown below in Table 3.Table 3. Liquid Phase Reagent Component Amount Units Water Fill to 1 L

[0093] The paramagnetic particles covalently labeled with monoclonal anti-CA 19-9 antibody were added to the 50 mL of CA 19-9 SP Buffer Compositions 1-5 from Table 2 to form five respective Solid Phase Reagents 1-5 with varying concentrations of MgSO4. Assay Description:

[0094] In a non-limiting embodiment, ADVIA Centaur CA 19-9 assay is a two-step sandwich immunoassay using direct chemiluminometric technology which uses a single monoclonal antibody, 1116-NS-19-9 (obtained from Fujirebio Diagnostics, Inc.) for both the solid phase reagent and the liquid phase reagent. The antibody was covalently coupled to paramagnetic particles for the solid phase reagent, and the same clone of antibody was labeled with acridinium ester in the liquid phase reagent. The system dispensed 75 µL of the sample into a cuvette, and then 4.75 minutes later, 350 µL of the solid phase reagent was added. This mixture was incubated at 37°C for 8.25 minutes followed by a wash step using ADVIA Centaur Wash 1 (phosphatebuffered saline with Tween 20 and sodium azide) to remove excess unbound antigens. After the first wash, 100 µL of ADVIA Centaur Wash 1 was added to resuspend the particles. Then 100 µL of liquid phase reagent was reacted with the solid phase-bound CA 19-9 antigens for an additional 18-minute incubation, where it is washed again with ADVIA Centaur Wash 1. Then 300 µL each of acid reagent (0.5% peroxide and 0.45% nitric acid) and base reagent (0.25 M NaOH, 0.45% w / v ARQUAD 16-50) were added to the cuvette to initiate the chemiluminescent reaction. A direct relationship exists between the concentration of CA 19-9 present in a patient sample and the amount of relative light units (RLUs) detected by the system.

[0095] A summary of the assay steps are shown below in Table 4. Table 4. Assay Method Step Action ) Results:

[0096] The CA 19-9 immunoassay was performed as described above using the liquid phase reagent of Table 4 with each of Solid Phase Reagents 1-5 on three biological samples, serum “SST”, lithium heparin plasma (“LiHep”) and EDTA plasma (“EDTA”) from the same patient. The results are shown below in Table 5.Table 5. Recovery of CA 19-9 CA 19-9 (U / mL)Normal Range 100 U / mL 300 U / mLA .0 .5 9 9 0 % % %, ma bias relative to serum was found when increasing amounts of MgSO4 were added to the solid phase reagent composition. Example 2- Atellica IM CA 19-9 II Assay Reagent Design and Syntheses:

[0098] In this Example 2, a conjugate, HEGAE-DiSFPD-Mab-19-9, was synthesized as described in International Patent Publication WO 2022 / 126055. All reagents were evaluated on the Siemens Atellica® IM analyzer using a sandwich assay format as illustrated in FIG.2.

[0099] The CA 19-9 immunoassay reagents in this Example 2 included one solid phase (SP) reagent and one liquid phase reagent (also referred to as “lite reagent”) (FIG. 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 the HEGAE-DiSFPD-Mab-19-9 conjugate. Both solid phase and liquid phase reagents form the sandwich assay. The synthesis of MAb-coated solid phase is a well-known and straight forward process, and therefore no further description thereof is deemed necessary. As such, this Example 2 focuses on the creation of new solid phase and liquid phase reagents and their performance in a CA 19-9 immunoassay. Thus, in the present Example 2, new solid phase reagents and liquid phase reagents were designed which can mitigate EDTA plasma bias. It was surprisingly found that a mitigation in EDTA plasma bias relative to serum samples can be achieved when including MgSO4 in the solid phase reagent composition. The reagents were prepared as follows.

[0100] A solid phase reagent composition was prepared with the composition shownbelow in Table 6. Table 6. Solid Phase Reagent Composition Component Amount Units Water Fill to 1 L

[0101] In Table 6, “PMP-MAb 19-9” refers to paramagnetic particles covalently labeled with monoclonal anti-CA 19-9.

[0102] A liquid phase reagent was prepared with the following composition shown belowin Table 7. Table 7. Liquid Phase Reagent Component Amount Units Water Fill to 1 LAssay Description:

[0103] In a non-limiting embodiment, Atellica IM CA 19-9 II assay is a two-step sandwich immunoassay using direct chemiluminometric technology which uses a single monoclonal antibody, 1116-NS-19-9 (obtained from Fujirebio Diagnostics, Inc.) for both the solid phase reagent and the liquid phase reagent. The antibody was covalently coupled to paramagnetic particles for the solid phase reagent, and the same clone of antibody was labeled with acridinium ester in the liquid phase reagent. The system dispensed 75 µL of the sample into a cuvette, and then 350 µL of the solid phase reagent was added. This mixture was incubated at 37°C for 12 minutes followed by a three separate washes using Atellica IM Wash (phosphate buffered saline with Tween 20 and sodium azide) to remove excess unbound antigens. After the third wash, 250 µL of water was added to resuspend the particles. Then 100 µL of liquid phase reagent was reactedwith the solid phase-bound CA 19-9 antigens for an additional 12-minute incubation, where it was washed again three separate times with Atellica IM Wash. Then 300 µL each of acid reagent (0.5% peroxide and 0.45% nitric acid) and base reagent (0.25 M NaOH, 0.45% w / v ARQUAD 16-50) were added to the cuvette to initiate the chemiluminescent reaction. A direct relationship exists between the concentration of CA 19-9 present in a patient sample and the amount of relative light units (RLUs) detected by the system.

[0104] A summary of the assay steps are shown below in Table 8. Table 8. Assay Method Step Action 1, ) 1,Results:

[0105] The CA 19-9 immunoassay was performed as described above using the solid phase reagent of Table 6 with the liquid phase reagent of Table 7. The results are shown below in Tables 9 and 10 and FIGS.3 and 4. Each sample is a matched serum and EDTA plasma sample drawn 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 of the EDTA plasma specimen tubes used for the samples. “K2” refers to dipotassium and the dipotassium (K2) EDTA plasma tubes have two potassium ions chelated to each EDTA molecule. “K3” refers to tripotassium and the tripotassium (K3) EDTA plasma tubes have three potassium ions chelated to each EDTA molecule. Functionally, the K2 EDTA or K3 EDTA acts as an anticoagulant by binding to calcium (Ca2+). The K3 EDTA tubes may also contain a preservative (e.g., a small amount of potassium oxalate or potassium fluoride). K2 EDTA tubes target 1.8 mg K2 EDTA per mL of blood, while the K3 EDTA tubes target 1.7 mg K3 EDTA per mL of blood. For example, a 10 mL K2 EDTA tube contains 18 mg of K2 EDTA. Using the ratio of the molecular weights of each component, this means that K2 EDTA tubes target roughly 0.35 mg of potassium per mL of blood and K3 EDTA tubes target roughly 0.45 mg of potassium per mL of blood.Table 9. Recovery of CA 19-9 Serum K2 EDTA CA 19-9 II Pla % Bias Sample sma Plasma vsTable 10. Recovery of CA 19-9 Serum K3 EDTA Plasma % Bias Sample CA 19-9 II CA 19-9 II Plasma vs

[0106] Tables 9 and 10 provide the CA 19-9 values measured for the matched serum and plasma (K2 EDTA in Table 9, K3 EDTA in Table 10) samples, which show very low mean bias between the serum and plasma recoveries. As shown in Tables 9 and 10, mitigation of EDTA plasma bias relative to serum occurred when using the solid phase reagent composition containing magnesium sulfate (MgSO4). NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0107] Illustrative embodiment 1. A solid phase reagent composition for an immunoassay for detecting cancer antigen 19-9 (CA 19-9) including: a solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof directly or indirectly attached thereto; and a divalent salt.

[0108] Illustrative embodiment 2. The solid phase reagent composition of illustrative embodiment 1, wherein the divalent salt is present in a concentration of greater than or equal to about 40 mM to less than or equal to about 60 mM.

[0109] Illustrative embodiment 3. The solid phase reagent composition of any one of the previous illustrative embodiments, wherein the divalent salt is MgSO4.

[0110] Illustrative embodiment 4. The solid phase reagent composition of any one of the previous illustrative embodiments, further including one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

[0111] Illustrative embodiment 5. The solid phase reagent composition of any one of the previous illustrative embodiments, further including ethylenediaminetetraacetic acid (EDTA) tetrasodium, sorbitol, heparin, and a monoclonal blocking antibody.

[0112] Illustrative embodiment 6. The solid phase reagent composition of any one of the previous illustrative embodiments, wherein the solid phase includes magnetic particles, paramagnetic particles, a membrane, latex particles, a tube, a microwell plate, a polystyrene bead, or nylon.

[0113] Illustrative embodiment 7. A liquid phase reagent composition for an immunoassay for detecting cancer antigen 19-9 (CA 19-9) including: a conjugate including: an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups; and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate, wherein the label is a dibromopyridazinedione-hexa(ethylene)glycol-dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I; and a divalent salt.

[0114] Illustrative embodiment 8. The liquid phase reagent composition of illustrative embodiment 7, wherein the divalent salt is present in a concentration of greater than or equal to about 1 mM to less than or equal to about 10 mM.

[0115] Illustrative embodiment 9. The liquid phase reagent composition of illustrativeembodiment 7 or 8, wherein the divalent salt is MgSO4.

[0116] Illustrative embodiment 10. The liquid phase reagent composition of any one of illustrative embodiments 7 to 9, further including one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

[0117] Illustrative embodiment 11. The liquid phase reagent composition of any one of illustrative embodiments 7 to 10, further including ethylenediaminetetraacetic acid (EDTA) tetrasodium and sorbitol.

[0118] Illustrative embodiment 12. An immunoassay kit for detecting cancer antigen 19- 9 (CA 19-9) including: a) a solid phase reagent including: a solid phase having an anti-CA 19-9 monoclonal antibody or fragment thereof directly or indirectly attached thereto; and a divalent salt; and b) a liquid phase reagent including: a conjugate including: an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups; and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate, wherein the label is a dibromopyridazinedione-hexa(ethylene)glycol-dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I.

[0119] Illustrative embodiment 13. The immunoassay kit of illustrative embodiment 12, wherein the divalent salt is present in a concentration of greater than or equal to about 40 mM to less than or equal to about 60 mM in the solid phase reagent.

[0120] Illustrative embodiment 14. The immunoassay kit of illustrative embodiment 12 or 13, wherein the liquid phase reagent further includes the divalent salt.

[0121] Illustrative embodiment 15. The immunoassay kit of any one of illustrative embodiments 12 to 14, wherein the divalent salt is present in a concentration of greater than or equal to about 1 mM to less than or equal to about 10 mM in the liquid phase reagent.

[0122] Illustrative embodiment 16. The immunoassay kit of any one of illustrativeembodiments 12 to 15, wherein the divalent salt is MgSO4.

[0123] Illustrative embodiment 17. The immunoassay kit of any one of illustrative embodiments 12 to 16, wherein the immunoassay kit further includes one or more of a wash solution, a dilution solution, an excipient, an interference solution, a positive control, a negative control, a calibration reagent, and a quality control reagent.

[0124] Illustrative embodiment 18. The immunoassay kit of any one of illustrative embodiments 12 to 17, where the solid phase reagent composition and the liquid phase reagent composition each further includes one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

[0125] Illustrative embodiment 19. The immunoassay kit of any one of illustrative embodiments 12 to 18, wherein the solid phase reagent composition further includes EDTA tetrasodium, sorbitol, heparin, and a monoclonal blocking antibody and the liquid phase reagent composition further includes EDTA tetrasodium and sorbitol.

[0126] Illustrative embodiment 20. The immunoassay kit of any one of illustrative embodiments 12 to 19, wherein the solid phase includes magnetic particles, paramagnetic particles, a membrane, latex particles, a tube, a microwell plate, a polystyrene bead, or nylon.

[0127] Illustrative embodiment 21. A method of detecting cancer antigen 19-9 (CA 19- 9) in a sample, the method includes: combining the sample with the solid phase reagent composition and the liquid phase reagent composition of the immunoassay kit of claim 12 to form a mixture; incubating the mixture under conditions that allow for binding of the solid phase reagent composition and the liquid phase reagent composition to CA 19-9 present in the sample, thereby forming a complex; and detecting the complex.

[0128] Illustrative embodiment 22. The method of illustrative embodiment 21, wherein the combining and the incubating includes: a) a first combining step including combining the sample with the solid phase reagent composition to produce a solid phase-containing mixture and incubating the solid phase-containing mixture for a suitable amount of time; and b) a second combining step including combining the solid phase-containing mixture with the liquid phase reagent composition to produce the mixture and incubating the mixture for a suitable amount of time.

[0129] Illustrative embodiment 23. The method of illustrative embodiment 22, further including a first separation step including separating the solid phase from the solid phase- containing mixture and a second separation step including separating the solid phase from the mixture.

[0130] Illustrative embodiment 24. The method of any one of illustrative embodiments21 to 23, wherein the sample includes plasma or serum.

[0131] Illustrative embodiment 25. The method of any one of illustrative embodiments 21 to 24, wherein the sample includes plasma.

[0132] Illustrative embodiment 26. The method of any one of illustrative embodiments 21 to 25, wherein at least one of the steps is performed on an automated analyzer.

[0133] Illustrative embodiment 27. A non-transitory computer readable medium containing executable instructions that when executed cause a processor to perform operations including the method of any one of illustrative embodiments 21 to 26.

[0134] Illustrative embodiment 28. The non-transitory computer readable medium of illustrative embodiment 27, wherein the instructions cause the processor to control an automated analyzer to execute at least one of the steps of the method.

[0135] 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

CLAIMS What is claimed is:

1. A solid phase reagent composition for an immunoassay for detecting 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 a divalent salt.

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

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

4. The solid phase reagent composition of any one of the previous claims, further comprising one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

5. The solid phase reagent composition of any one of the previous claims, further comprising ethylenediaminetetraacetic acid (EDTA) tetrasodium, sorbitol, heparin, and a monoclonal blocking antibody.

6. The solid phase reagent composition of any one of the previous claims, wherein the solid phase comprises magnetic particles, paramagnetic particles, a membrane, latex particles, a tube, a microwell plate, a polystyrene bead, or nylon.

7. A liquid phase reagent composition for an immunoassay for detecting cancer antigen 19- 9 (CA 19-9) comprising: a conjugate comprising: an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups; and at least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate, wherein the label is a dibromopyridazinedione-hexa(ethylene)glycol-dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I; and a divalent salt.

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

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

10. The liquid phase reagent composition of any one of claims 7 to 9, further comprising one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

11. The liquid phase reagent composition of any one of claims 7 to 10, further comprising ethylenediaminetetraacetic acid (EDTA) tetrasodium 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 a divalent salt; and b) a liquid phase reagent comprising: a conjugate comprising: an anti-CA 19-9 monoclonal antibody or fragment thereof having at least one disulfide bond capable of reduction to two sulfhydryl groups; andat least one label directly or indirectly attached to the two sulfhydryl groups of the anti-CA 19-9 monoclonal antibody or fragment thereof, thereby forming the conjugate, wherein the label is a dibromopyridazinedione-hexa(ethylene)glycol- dimethylphenyl acridinium ester (DiBrPD-HEGAE) having the structure of Formula I:Formula I.

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

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

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

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

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

18. The immunoassay kit of any one of claims 12 to 17, where the solid phase reagent composition and the liquid phase reagent composition each further comprises one or more of an excipient, a sugar alcohol, a buffer, an antibiotic, or a surfactant.

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

20. The immunoassay kit of any one of claims 12 to 19, wherein the solid phase comprises magnetic particles, paramagnetic particles, a membrane, latex particles, a tube, a microwell plate, a polystyrene bead, or nylon.

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

22. The method of claim 21, wherein the combining and the incubating comprises: a) a first combining step comprising combining the sample with the solid phase reagent composition to produce a solid phase-containing mixture and incubating the solid phase- containing mixture for a suitable amount of time; and b) a second combining step comprising combining the solid phase-containing mixture with the liquid phase reagent composition to produce the mixture and incubating the mixture for a suitable amount of time.

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

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

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

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

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

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

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