Antibodies against methotrexate and their uses

Abstract

This specification discloses antibodies that specifically bind to methotrexate analyte. Such antibodies can be used to detect methotrexate analyte in samples in homogeneous enzyme immunoassays and the like. Hapten structures for inducing such antibodies are also described. Furthermore, conjugates useful for immunoassays using such antibodies are also described.

Description

[Technical Field]

[0001] [Reference to related applications] This application claims priority under U.S. Patent Application No. 18 / 186,899, filed on 20 March 2023, the disclosures of which are incorporated herein by reference.

[0002] [Technical field to which the review belongs] This invention relates to a method and system for detecting methotrexate (MTX) using an immunoassay. In particular, it relates to antibodies and haptens used in an immunoassay for detecting and quantifying MTX in a biological sample.

[0003] [Inclusion via array listing reference] The Sequence Listing (SEQ) was created on March 20, 2023, and is provided to this application as a Sequence Listing XML file named "ARKD-009_SEQ_LIST.xml" with a size of 35,274 bytes. The contents of the Sequence Listing XML are incorporated into this application by reference. [Background technology]

[0004] MTX[N-[4[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, abbreviated as MTX], formerly known as ametopterin, is an antimetabolite used to treat certain neoplastic diseases, severe psoriasis, and adult rheumatoid arthritis.

[0005] MTX has a unique mechanism of action for use in chemotherapy and immunosuppression in autoimmune diseases. In cancer treatment, MTX acts as an anti-folate anti-metabolite. MTX is taken up into cells by a carrier called the human reduced folate carrier, forming MTX-polyglutamate. Both MTX and MTX-polyglutamate inhibit dihydrofolate reductase, an enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, the active form of folate. Tetrahydrofolate is essential for the synthesis of both DNA and RNA nucleotides. MTX-polyglutamate further inhibits the de novo synthesis of purines by both purine synthase and thymidylate synthase, thereby inhibiting DNA synthesis. This mechanism of action is utilized in cancer treatment due to its cytotoxic effects.

[0006] In autoimmune diseases, different mechanisms of action are involved when MTX is selected as a drug of choice. MTX inhibits the enzyme transformylase, which in turn disrupts adenosine and guanine metabolism. The accumulation of adenosine is converted into its anti-inflammatory effect, manifesting as suppression of T cell activation, downregulation of B cells, and increased sensitivity of activated CD-95 T cells. Furthermore, it manifests as suppression of methyltransferase activity and inhibition of interleukin-β1 binding to cell surface receptors.

[0007] MTX can have serious toxicity. Patients receiving MTX therapy must be closely monitored to ensure prompt detection of toxic effects. Moderate to high doses (approximately 35 mg / m²) 2 ~12g / m 2 The combination of MTX and leucovorin (citroborum factor) rescue has shown favorable results in the treatment of osteosarcoma, leukemia, non-Hodgkin lymphoma, lung cancer, and breast cancer.

[0008] High-dose MTX (HDMTX) refers to doses exceeding 500 mg / mL. Patients may experience nausea, mucosal ulcers, hair loss, fatigue, fever, increased risk of infection, leukopenia, gastrointestinal bleeding, pancreatitis, cirrhosis, aplastic anemia, malignant tumors (lymphoproliferative disorders), infections, interstitial pneumonia, renal dysfunction, and teratogenicity. Accurate monitoring of MTX blood levels is crucial to ensure safe and effective treatment and to avoid toxicity from excessive exposure.

[0009] Three antidotes used for MTX toxicity are leucovorin, thymidine, and glucarpidase. Leucovorin is the reduced active form of folic acid and rescues normal cells from toxic effects caused by MTX inhibition of reduced folic acid. Leucovorin is particularly effective in preventing myelosuppression, gastrointestinal toxicity, and neurotoxicity during MTX treatment. Thymidine rescues cells from the cytotoxic effects of MTX, but its use is still in the research stage and is always used in combination with other drugs. Glucarpidase converts MTX into non-toxic metabolites, 4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzoic acid (DAMPA) and glutamic acid, and rapidly removes MTX from patients with renal impairment. Glucarpidase, in combination with leucovorin, is a common treatment for MTX toxicity. A single dose of glucarpidase reduces plasma MTX concentration by more than 97% within 15 minutes. For renal dysfunction, hydration and alkalinization of the urine are recommended.

[0010] Serum concentrations of MTX depend on the indication for use, dosage, route of administration, treatment regimen, individual pharmacokinetics, metabolism, and other clinical factors. For example, in breast cancer treatment, serum concentrations usually reach 10-100 μmol / L, but in high-dose therapy for osteosarcoma, they can exceed 1000 μmol / L, and in some cases, pediatric osteosarcoma patients have reached 3100 μmol / L after 4 hours of intravenous administration. The decay curve of MTX in osteosarcoma treatment shows wide variation, with concentrations of 30-300 μmol / L at 24 hours, 3-30 μmol / L at 48 hours, and less than 0.3 μmol / L at 72 hours. Typically, 10 mg of leucovorin is administered intravenously 24 hours after the start of MTX infusion. Subsequent doses are adjusted based on the MTX concentrations achieved at 24, 48, and 72 hours. MTX concentrations exceeding 50 μmol / L after 24 hours, 10 μmol / L after 48 hours, and 0.5 μmol / L after 72 hours suggest potential toxicity, and treatment is usually continued by increasing the leucovorin dose according to an algorithm until the MTX concentration falls below 0.1 μmol / L. Guidelines for MTX therapy with leucovorin rescue generally recommend continuing leucovorin until the MTX concentration falls below 0.05 μmol / L. Therefore, there is a need for analytical methods to determine MTX concentrations in patient samples, particularly plasma and serum, with high sensitivity and specificity.

[0011] Several analytical methods are used to determine the concentration of MTX in serum or plasma.

[0012] To determine MTX concentration at varying levels of sensitivity and specificity, the following analytical methods have been used: fluorescence-polarized immunoassay (FPIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS / MS), and enzyme amplification immunoassay (EMIT).

[0013] Homogeneous immunoassays have been used in clinical laboratories and hospitals for over 40 years to monitor the serum or plasma concentrations of therapeutic drugs. Advantages of immunoassays include accuracy, high sensitivity, and ease of operation in many commercially available assay formats. Immunoassays for measuring MTX are commercially available, and their availability allows for the routine measurement of drug concentrations in patient samples. However, these assays have some limitations in terms of sensitivity and specificity. Immunoassays rely on the selection of antibody reagents to provide specificity, which is usually challenging because constructing drug analogs suitable for binding to large molecules (such as proteins) is typically difficult or impossible in order to develop immunogens that induce antibodies that react with the drug. Often, the derivatization required to produce the immunogen significantly alters the drug, resulting in antibodies that recognize the analog but not the drug. Therefore, developing immunoassays requires preparing analogs that are suitable for binding to proteins and induce antibodies that recognize both the analog and the drug. The IUPAC nomenclature name for MTX is (2S)-2-[[4-[(2,4-diaminopteridine-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid, also known as N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid (CAS number 59-05-2).

[0014] The structure and IUPAC name of MTX are also shown in Figure 1.

[0015] Abbott has commercialized an FPIA-based assay for MTX on TDx / FLx, but its throughput has been limited due to the complexity of the optical configuration for capturing the signal and the difficulty in integrating it with other assays. The principle of FPIA (fluorescence polarization immunoassay) is based on the competitive binding of a small molecule (free antigen) to an antibody and a fluorescein-labeled antigen. As the amount of antigen increases, the amount of labeled antigen that binds to the antibody decreases, resulting in a detection response that is inversely proportional to the antigen concentration. The signal of this assay is polarized light, and its intensity decreases with increasing antigen concentration, and is not based on kinetic reading. Such embodiments of the present invention utilize a kinetic reading in which the signal is directly proportional to the analyte concentration.

[0016] Siemens' Syva® EMIT® technology utilizes the principle of competitive enzyme immunoassay based on competition between drugs in a sample and MTX labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for binding to an antibody reagent. When the latter binds to the antibody, enzyme activity decreases. If a drug is present in the sample, enzyme activity increases, and its magnitude is directly proportional to the drug concentration. The active enzyme converts the coenzyme nicotinamide adenine dinucleotide (NAD+) to NADH, a conversion measured by spectrophotometrics as a percentage change in absorbance. Endogenous serum G6PDH is equivalent to the coenzyme NAD+. + Because it functions only with the bacterial enzyme used in this assay, it does not interfere with the measurement results. However, the sensitivity of this assay is limited, and the product description states it is 0.3 mmol / L (Mendu, DRet. al. 2007, Ther. Drug. Monit 29:632-637). Therefore, a more sensitive assay is needed.

[0017] To create an immunoassay that is specific to MTX and does not cross-react with its major metabolite, 7-OH MTX, the antibody is directed to target the pteridine ring portion of the molecule.

[0018] U.S. Patent No. 11,054,430, "Compounds and Methods for MTX Detection," describes compounds and methods for detecting MTX in biological samples using MTX-specific antibodies with minimal cross-reactivity to 7-OH MTX and DAMPA. However, this assay exhibits high cross-reactivity to 7-OH MTX and has a limited dynamic range. Therefore, there is a need for better specificity and a wider dynamic range. [Overview of the project] [Problems that the invention aims to solve]

[0019] This disclosure provides immunoassays for MTX analytes. In some embodiments, this disclosure relates to the use of MTX hapten derivatives, tracers, and conjugates in signaling immunoassay systems. This disclosure also relates to the use of MTX analog immunogens used to produce antibodies that capture such analytes. [Means for solving the problem]

[0020] In some embodiments, the disclosure provides MTX derivatives that are acylated and / or alkylated on the phenyl group of MTX (see Figure 1). In certain embodiments, such derivatives are used to create conjugates useful for immunoassays described herein.

[0021] In some embodiments, the disclosure provides polyclonal or monoclonal antibodies that specifically bind to MTX and exhibit less than 0.1% cross-reactivity to 7-hydroxyMTX metabolites in the presence of the parent compound.

[0022] In some embodiments, the disclosure provides polyclonal or monoclonal antibodies that specifically bind to MTX.

[0023] In some embodiments, the antibody may specifically bind to one or more of MTX, pralatrexate, or phototrexate.

[0024] In some embodiments, the disclosure provides polyclonal or monoclonal antibodies that specifically bind to free MTX and exhibit less than 0.1% cross-reactivity to triamterene and trimethoprim.

[0025] In some embodiments, the disclosure provides methods for synthesizing haptens, immunogens, and conjugates using 2,4-diaminopteridine as a starting material. In some embodiments, the synthesis involves attaching a linking group to a protein or label (e.g., a labeling enzyme) via the phenyl group of 2,4-diamino-6-pteridinylmethylaminomethyl (DAM).

[0026] This disclosure relates to novel haptens derived from the phenyl moiety of diaminopteridinylbenzoic acid. In some embodiments, these haptens are conjugated to proteins via various linkers, thereby providing monoclonal antibody and enzyme conjugates that further reduce cross-reactivity.

[0027] Furthermore, this disclosure provides antibodies having binding specificity to MTX. In some cases, the MTX antibody comprises a variable heavy chain and a variable light chain, wherein the amino acid sequence of the heavy chain comprises SEQ ID NO: 1 and the amino acid sequence of the light chain comprises SEQ ID NO: 5.

[0028] This disclosure also describes an antibody having binding specificity to MTX, wherein the heavy chain variable region and the light chain variable region are antibodies, the amino acid sequence of the heavy chain variable region includes SEQ ID NO: 9, and the amino acid sequence of the light chain variable region includes SEQ ID NO: 13.

[0029] This disclosure relates to an antibody that generally has binding specificity to MTX and includes a heavy chain variable region and a light chain variable region, wherein the nucleotide sequence of the heavy chain variable region includes SEQ ID NO: 17 and the nucleotide sequence of the light chain variable region includes SEQ ID NO: 21.

[0030] The Disclosure further describes a method for detecting MTX in a sample, the method comprising the step of mixing a capture molecule and labeled MTX with the sample in solution, wherein the capture molecule is capable of binding to the labeled MTX, and the MTX present in the sample competes with the labeled MTX for binding to the capture molecule. The method comprises detecting the amount of labeled MTX bound to the capture molecule via a signal generated by the label on the bound labeled MTX, the signal being inversely proportional to the amount of MTX present in the sample.

[0031] Furthermore, the present disclosure provides a kit for detecting MTX in a sample. The kit comprises a capture molecule and labeled MTX, wherein the capture molecule is bindable to the labeled MTX, and the MTX competitively inhibits the binding of the labeled MTX to the capture molecule. The kit may also include instructions for performing a detection assay, which include mixing the sample with the capture molecule and labeled MTX in solution (wherein the capture molecule is bindable to the labeled MTX, and the MTX present in the sample competes with the labeled MTX for binding to the capture molecule); and detecting the amount of labeled MTX bound to the capture molecule via a signal generated by the label on the bound labeled MTX (wherein the signal is inversely proportional to the amount of MTX present in the sample); [Brief explanation of the drawing]

[0032] This disclosure can be better understood from the following detailed description made in conjunction with the attached drawings that form part of this application. [Figure 1] The chemical structure of MTX and its numbering system according to the Alternative Nomenclature (IUPAC) are shown. [Figure 2]The chemical structures of the metabolites DAMPA and 7-OH-MTX are shown. [Figure 3] The chemical structure of the MTX analog is shown. [Figure 4] The synthesis scheme for DAMP-1 hapten according to the embodiments of this disclosure is shown. [Figure 5] The synthesis scheme for DAMP-2, an MTX analog hapten according to the embodiments of this disclosure, is shown. [Figure 6] The synthesis scheme for DAMP-3 hapten according to the embodiments of this disclosure is shown. [Figure 7] The synthesis scheme for DAMP-4 hapten according to the embodiments of this disclosure is shown. [Figure 8] The synthesis scheme for DAMP-5 hapten according to the embodiments of this disclosure is shown. [Figure 9] The synthesis scheme for DAMP-6, an MTX analog hapten according to the embodiments of this disclosure, is shown. [Figure 10] This disclosure shows a synthesis scheme for DAMP-YS-G6PDH according to one embodiment. [Figure 11] This disclosure shows a synthesis scheme for a DAMP-YS-KLH immunogen according to one embodiment. [Figure 12] The synthesis scheme for the DAMP-YS-BSA immunogen according to the embodiments of this disclosure is shown. [Figure 13] The principles of homogeneous enzyme immunoassay and antibody screening technologies according to embodiments of this disclosure are shown. [Figure 14] The graph shows an MTX calibration curve using the monoclonal antibody 64H1-K1 according to the embodiment of this disclosure. [Figure 15] A scatter plot using Passing & Bablok fit is shown comparing the MTX assay and LC-MS / MS described herein.

[0033] (A brief explanation of sequence listings) Sequence ID 1: Amino acid sequence of the heavy chain variable region of antibody clone 28 of this disclosure SEQ ID NO: 2 Amino acid sequence of the first complementarity-determining region (CDR1) of the heavy chain variable region of SEQ ID NO: 1 SEQ ID NO: 3 Amino acid sequence of the second complementarity-determining region (CDR2) of the heavy chain variable region of SEQ ID NO: 1 SEQ ID NO: 4 Amino acid sequence of the third complementarity-determining region (CDR3) of the heavy chain variable region of SEQ ID NO: 1 Sequence ID 5: Amino acid sequence of the light chain variable region of antibody clone 28 of the present disclosure SEQ ID NO: 6 Amino acid sequence of CDR1 in the light chain variable region of SEQ ID NO: 5 SEQ ID NO: 7 Amino acid sequence of CDR2 in the light chain variable region of SEQ ID NO: 5 SEQ ID NO: 8 Amino acid sequence of CDR3 in the light chain variable region of SEQ ID NO: 5 SEQ ID NO: 9 Amino acid sequence of the heavy chain variable region of antibody clone 32 of the present disclosure SEQ ID NO: 10 Amino acid sequence of CDR1 in the heavy chain variable region of SEQ ID NO: 9 SEQ ID NO: 11 Amino acid sequence of CDR2 in the heavy chain variable region of SEQ ID NO: 9 SEQ ID NO: 12 Amino acid sequence of CDR3 in the heavy chain variable region of SEQ ID NO: 9 Sequence ID No. 13: Amino acid sequence of the light chain variable region of antibody clone 32 of the present disclosure SEQ ID NO: 14 Amino acid sequence of CDR1 in the light chain variable region of SEQ ID NO: 13 SEQ ID NO: 15 Amino acid sequence of CDR2 in the light chain variable region of SEQ ID NO: 13 SEQ ID NO: 16 Amino acid sequence of CDR3 in the light chain variable region of SEQ ID NO: 13 Sequence ID No. 17 Amino acid sequence of the heavy chain variable region of antibody clone 64 of this disclosure SEQ ID NO: 18 Amino acid sequence of CDR1 in the heavy chain variable region of SEQ ID NO: 17 SEQ ID NO: 19 Amino acid sequence of CDR2 in the heavy chain variable region of SEQ ID NO: 17 SEQ ID NO: 20 Amino acid sequence of CDR3 in the heavy chain variable region of SEQ ID NO: 17 Sequence ID No. 21: Amino acid sequence of the light chain variable region of antibody clone 64 of this disclosure SEQ ID NO: 22 Amino acid sequence of CDR1 in the light chain variable region of SEQ ID NO: 21 SEQ ID NO: 23 Amino acid sequence of CDR2 in the light chain variable region of SEQ ID NO: 21 SEQ ID NO: 24 Amino acid sequence of CDR3 in the light chain variable region of SEQ ID NO: 21 Sequence ID 25: Nucleotide sequence of a representative nucleic acid encoding the heavy chain variable region of Sequence ID 1. Sequence ID 26: A representative nucleic acid nucleotide sequence encoding the light chain variable region of Sequence ID 5. Sequence ID 27: A representative nucleic acid nucleotide sequence encoding the heavy chain variable region of Sequence ID 9. Sequence ID 28: A representative nucleic acid nucleotide sequence encoding the light chain variable region of Sequence ID 13. Sequence ID 29: A representative nucleic acid nucleotide sequence encoding the heavy chain variable region of Sequence ID 17. Sequence ID 30 is a representative nucleic acid nucleotide sequence encoding the light chain variable region of Sequence ID 21. Before describing the present invention in further detail, it should be understood that the present invention is not limited to the specific embodiments described and, of course, various modifications are possible. Furthermore, the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. The scope of the present invention is limited only by the appended claims.

[0034] Where a numerical range is specified, each intermediate value between the upper and lower limits of that range (including values ​​in increments of one-tenth of the lower limit unit, unless otherwise clearly indicated in the context) and any other express or intervening values ​​within that range are understood to be included in the invention. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are included in the invention unless there are limits specifically excluded in the stated range. If the stated range includes one or both limits, the range excluding one or both of those limits is also included in the invention. The exact numerical values ​​used in the specification and claims constitute additional embodiments of the invention and are intended to encompass any range narrowed between any two endpoints within the provided exemplary ranges and values. Efforts have been made to ensure the accuracy of the numerical values ​​disclosed herein. However, any measurement may inherently contain some error due to the standard deviation inherent in the measurement technique.

[0035] Certain features of the present invention are described in the context of individual embodiments for clarity, but it is understood that they may also be provided in combination in a single embodiment. Conversely, various features of the present invention are described in the context of a single embodiment for brevity, but may also be provided individually or in any suitable partial combination. All combinations of embodiments according to the present invention are considered individually and expressly disclosed herein and are particularly included in the present invention, insofar as they encompass subject matter such as stable compounds (i.e., compounds that can be manufactured, isolated, characterized, and tested for biological activity). Furthermore, any partial combinations of various embodiments and their elements (e.g., elements of chemical groups enumerated in the embodiments describing the variables) are also considered individually and expressly disclosed herein and are particularly included in the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. While methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference to disclose and explain the methods and / or materials cited herein. Supplemental information published with such publications, patents, and patent applications is also incorporated herein by reference. For example, some academic papers are accompanied by supplemental information that is usually available online.

[0037] In this specification and the attached claims, singular nouns (a, an, the) also refer to multiple subjects unless the context clearly indicates otherwise. Furthermore, it should be noted that claims may be constructed to exclude any optional element. Therefore, this statement is intended to serve as an antecedent basis for the use of exclusive terms such as "solely" or "only," or for the use of "negative" limitations, in relation to the enumeration of elements of the claims.

[0038] Certain features of the present invention are described in the context of individual embodiments for clarity, but it is understood that they may also be provided in combination in a single embodiment. Conversely, various features of the present invention are described in the context of a single embodiment for simplification, but they may also be provided individually or in any suitable partial combination.

[0039] The publications, patents, and patent applications referenced herein are provided solely for disclosure prior to the filing date of this application. This description should not be construed as acknowledging that the present invention has no prior rights to such publications based on prior invention. Note that the publication dates mentioned may differ from the actual publication dates, in which case separate verification is required. [Modes for carrying out the invention]

[0040] Before proceeding further with a description of specific embodiments of this disclosure, we define some terms.

[0041] Analyte An analyte is a compound or composition to be determined, and is a substance of interest. An analyte is one member of a specific binding pair (SBP), can be monovalent or polyvalent ligand, is typically antigenic or haptenic, and is a single or multiple compounds that share at least one common epitope or determining site.

[0042] MTX Analyte In this specification, "MTX analyte" refers to an analyte having an antibody-binding epitope common to MTX. The analytes contained in "MTX analyte" include methotrexate (MTX) and exhibit low cross-reactivity with 7-hydroxy MTX (7-OH MTX) and similar pteridinil-containing analogs.

[0043] Sample suspected of containing analyte The method of this disclosure allows for the analysis of any sample that is reasonably presumed to contain an analyte. Such samples may include human, animal, or artificial samples. The sample may be prepared in any suitable medium that does not interfere with the assay. Typically, the sample may be an aqueous solution or a naturally occurring bodily fluid, such as urine, whole blood, serum, plasma, cerebrospinal fluid, or saliva. In some cases, the sample is serum.

[0044] Measuring the amount of analyte Quantitative, semi-quantitative, and qualitative methods, as well as all other methods for measuring analytes, are considered to be included in methods for measuring the amount of analytes. For example, a method for simply detecting the presence or absence of analytes in a sample that is presumed to contain analytes is also considered to be within the scope of this disclosure.

[0045] Synonyms for the expression “measurement of the amount of analyte” that are considered to be included in the scope of this disclosure include, but are not limited to, the detection, measurement, or determination of analyte; the detection, measurement, or determination of the presence of analyte; the detection or determination of the amount of analyte; and the detection, measurement, or determination of the concentration of analyte.

[0046] Member of a Specific Binding Pair A member of a specific binding pair (SBP member) is one of two distinct molecules that has a region on its surface or within its cavity that specifically forms a binding complementary to a particular steric and polar configuration of the other molecule. Members of a specific binding pair are also referred to as ligands and receptors (antiligands), SBP members, and SBP partners. These are typically members of immunological pairs, such as antigen-antibody pairs.

[0047] Ligand An analyte is any organic compound whose receptor is naturally occurring or can be prepared. For example, in a certain context of this disclosure, an analyte is a ligand, and this disclosure provides a method for determining the amount or concentration of an analyte that is a ligand.

[0048] Receptor A receptor is any compound or composition that can recognize a specific steric and polar configuration of a molecule. Such an organized region in a molecule is called an epitope or determinant site. Examples of naturally occurring receptors include antibodies and enzymes.

[0049] Epitope An "epitope" refers to a molecular region on the surface of an antigen that triggers an immune response and can bind to specific antibodies produced by that response. It is also called a "determinant" or "antigenic determinant." In the case of haptens (e.g., MTX), antibodies against non-antigenic hapten molecules can be produced by conjugating the hapten to an immunogenic carrier. At this time, antibodies that recognize the "epitope" defined by the hapten are produced.

[0050] Linking Group A linking group is a structural component that connects two or more substructures. A linking group has at least one continuous atomic chain extending between the substructures. The atoms constituting the linking group are linked to each other by chemical bonds. The number of atoms in a linking group is determined by counting the atoms other than hydrogen.

[0051] Conjugate A conjugate is a molecule in which two or more substructures are linked together via a linking group to form a single structure. The linking is achieved by linking the substructures via the linking group. In the context of this disclosure, conjugates may include glucose-6-phosphate dehydrogenase (G6PDH) enzymes linked to haptens, SBP members, or analyte analogs, such as conjugates using G6PDH mutant enzymes (e.g., recombinant G6PDH as described in U.S. Patents 6,455,288, 6,090,567, and 6,033,890). In the context of this disclosure, G6PDH may also be referred to as an enzyme such as the G6PDH enzyme, or as a label such as a G6PDH label. In some cases, the conjugate may include, but is not limited to, labels such as G6PDH, alkaline phosphatase, β-galactosidase, horseradish peroxidase, or chemical labels such as fluorescent, luminescent, or colorimetric molecules conjugated to haptens, SBP members, or analyte analogs.

[0052] Conjugation Conjugation refers to any process in which two subunits are linked to each other to form a conjugate. A conjugation process may include any number of steps, as described herein, for example.

[0053] Hapten Haptens are capable of specifically binding to their corresponding antibodies, but they do not typically function as immunogens themselves for preparing those antibodies. By immunizing a hapten with a compound linked to an immunogenic carrier, antibodies that recognize that hapten can be prepared.

[0054] Derivative The term "derivative" refers to a chemical compound or molecule produced from a parent compound (parent compound) through one or more chemical reactions.

[0055] Analog The term "analog" refers to a compound that has a similar structure to another compound but differs in certain components. An analog may differ from the original compound by having one or more atoms, functional groups, or substructures replaced by other atoms, groups, or substructures.

[0056] Label "Label," "detection molecule," "reporter," or "detectable marker" refers to any molecule that generates or can generate a detectable signal. Labels can be conjugated to analytes, immunogens, antibodies, or receptors or molecules capable of binding to receptors (e.g., ligands, particularly haptens and antibodies). Labels can be conjugated directly or indirectly via linking groups. Non-limiting examples of labels include radioisotopes (e.g., 125 I) Examples include enzymes (e.g., β-galactosidase, peroxidase), G6PDH (e.g., mutant G6PDH such as recombinant G6PDH described in U.S. Patents 6,455,288, 6,090,567 and 6,033,890), enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores (e.g., rhodamine, fluorescein isothiocyanate, or FITC, Dylight 649), dyes, chemiluminescent substances and luminescent substances (e.g., dioxetane, luciferin), or sensitizers.

[0057] Immunogen The term "immunogen" refers to a substance that can induce, produce, or generate an immune response in a living organism.

[0058] Immunogenic carrier As used herein, "immunogenic carrier" refers to an immunogenic substance that can bind to a hapten at one or more sites, thereby enabling the production of antibodies that specifically bind to that hapten; generally, this is a protein. Examples of immunogenic carrier substances include, but are not limited to, proteins, glycoproteins, complex polyaminopolysaccharides, particles, and nucleic acids. These trigger an immune response from the host when recognized as exogenous. Polyaminopolysaccharides can be prepared from polysaccharides using conventional methods well known for this preparation.

[0059] Protein In this specification, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably to refer to polymeric forms of amino acids of any length. Unless otherwise specified, “polypeptide,” “peptide,” and “protein” may include genetically encoded and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, polypeptides having a modified peptide backbone, and fusion proteins.

[0060] Signal Producing System A "signal generation system" is used in an analyte assay and has one or more components, at least one of which is a detectable label (e.g., G6PDH such as mutant G6PDH). The signal generation system generates a signal related to the presence or amount of analyte in the sample. The signal generation system includes all reagents necessary to generate a measurable signal. For the purposes of this disclosure, typically, G6PDH or a labeled protein (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase) is conjugated to an SBP member similar to the analyte.

[0061] Other components of the signaling system include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding agents required for the binding of signaling substances, coenzymes, substances that react with enzyme products, other enzymes, and catalysts.

[0062] The signal generation system provides a signal detectable by external means such as measurement of electromagnetic radiation (e.g., visual observation). In some cases, the signal generation system includes a chromogenic substrate and an enzymatic label (e.g., a G6PDH mutant enzyme), the chromogenic substrate being enzymatically converted into a dye that absorbs ultraviolet or visible light.

[0063] Isolated In the context of antibodies, the term "isolated" means that the substance has been "artificially" altered from its natural state. That is, even if it is naturally occurring, it means that it has been altered from its original environment, removed, or both. For example, naturally occurring antibodies present in a living organism are not "isolated," but the same antibody separated from substances coexisting in that natural state is, in the sense of the term used herein, an "isolated" antibody. Antibodies may also be present in compositions that do not exist naturally, such as immunoassay reagents, and antibodies in such compositions are also considered isolated antibodies in the sense of the term used herein.

[0064] Interference "Interference" refers to a situation where a substance or process incorrectly affects the assay results. Interference can be endogenous or exogenous. Endogenous interference can be caused by substances present in the patient's sample. Exogenous interference can be caused by substances introduced into the patient's sample. Interference may be caused by hemolysis, jaundice, and dyslipidemia.

[0065] Drug interference can be caused by the parent drug, its metabolites, or additives in the drug formulation.

[0066] Pre-analytical interference may be due to the components of the sampling tube, transportation, and storage, which affect the determination of the analyte.

[0067] Carryover interference generally occurs when analytes from high-concentration samples (or reagents) are not completely removed by the cleaning process of the analytical system (cleaning of probes, mixers, or cuvettes).

[0068] Error rate (interference rate) (%) TIFF2026514355000002.tif20156

[0069] Cross-reactivity "Cross-reactivity" refers to the reaction of an antibody to an antigen that was not used to induce the antibody in question. Cross-reactivity can be determined in quantitative immunoassays by establishing a standard curve using known dilutions of the target analyte. Then, using this standard curve, the apparent concentration of the interfering substance present in known amounts in samples assayed under similar conditions is calculated. Cross-reactivity is obtained by dividing the apparent concentration by the actual concentration and multiplying the result by 100.

[0070] Cross-reactivity rate (%) TIFF2026514355000003.tif22156

[0071] Calibration and control materials The term "calibration material and control material" refers to any standard or reference material containing a known amount of analyte. Samples presumed to contain analyte and the corresponding calibration material are assayed under similar conditions. The concentration of analyte is calculated by comparing the results obtained from an unknown sample or specimen containing a known concentration of analyte with the results obtained from the standard material. This is usually done by constructing or creating a calibration curve.

[0072] Sensitivity "Sensitivity" is used in the sense of detection limit, that is, the minimum amount of analyte that produces a signal indistinguishable from the signal obtained when no analyte is present.

[0073] Spike Recovery Rate A "spike-recovery" assay refers to an assay that compares a known amount of analyte added (spiked) to a sample mixture with the amount of analyte measured in the sample mixture (recovery). The measured amount of analyte can be expressed as a concentration (ng / mL) or a percentage (%).

[0074] Substantial change in enzyme activity This refers to a change in enzyme activity sufficient to enable detection of an analyte when the enzyme is used as a label in an analyte assay. Typically, enzyme activity decreases by 10-100%, for example, 20-99%, or 30-95%.

[0075] Inhibitory antibody Inhibitory antibodies are antibodies that have the ability to inhibit the activity of an enzyme or an enzyme-ligand conjugate by binding to an epitope present on the enzyme. Such antibodies are distinguished from anti-ligand antibodies, which inhibit the enzymatic activity of an enzyme-ligand conjugate by binding to the ligand.

[0076] Modulation In assay experiments, "modulation" refers to the competition between haptens or analytes attached to labels such as enzymes and analytes in a sample presumed to contain analytes for the binding site of the analyte-antibody, resulting in a modulation of the amount of enzyme product produced (see Figure 10).

[0077] Maximum Inhibition "Maximum inhibition" refers to an antibody that, when an excess of the antibody is added to the assay and a signal is obtained in the absence of analytes, can bind to an epitope on the enzyme and inhibit the activity of the enzyme or enzyme-ligand conjugate.

[0078] Ancillary Materials Various auxiliary materials are frequently used in the assays relating to this disclosure. For example, the assay medium usually contains a buffer, as well as stabilizers for the assay medium and assay components. Often, in addition to these additives, additional proteins such as albumin, surfactants (especially nonionic surfactants), and binding promoters (e.g., polyalkylene glycols) may be included.

[0079] In certain embodiments, this disclosure provides anti-MTX antibodies induced using the immunogen of the present invention. Such antibodies can be used for the detection of MTX in immunoassays.

[0080] The MTX immunoassay described herein is based on the competition between MTX in a sample and a tracer, which is a labeled MTX analog of the herein, for binding to an anti-MTX antibody. In one embodiment, the immunoassay is a homogeneous enzyme immunoassay. Furthermore, an immunoassay kit is also provided.

[0081] The MTX analog according to the present invention is MTX derivatized to include a chemical moiety that facilitates the attachment of a carrier or label to the phenyl group of MTX, the phenyl group typically having a glutamic acid residue attached to the aminomethylaminobenzoyl moiety bonded to the 2,4-diaminopteridine moiety. The MTX analog of the present invention is derivatized at the phenyl group via a linking group substituted at the ortho, meta, or para position relative to N-10Me. The structure of MTX, showing the carbon number and the position of the benzoyl substituent, is shown, for example, in Figure 1.

[0082] As described in U.S. Patent No. 11,054,430, derivatizing the phenyl ring of the benzoyl moiety rather than the N-10Me group is advantageous because the moiety of the MTX analog available for antibody induction and recognition is detached from the MTX metabolite 7-hydroxy-MTX (7-OH MTX shown in Figure 2). Therefore, by conjugating the carrier via a linking group through the phenyl or benzoyl moiety of MTX, an immunogen that induces antibodies with minimal cross-reactivity with 7-hydroxy-MTX can be obtained.

[0083] By derivatizing the phenyl moiety while retaining the diaminopteridine nucleus, derivatives and haptens are obtained, which are immunologically sufficiently similar to MTX; therefore, antibodies induced by these analogs react with both the analog and MTX. Thus, the MTX analogs of the present invention, including a carrier, can induce anti-MTX antibodies. Furthermore, these MTX analogs can be labeled for use as tracers in immunoassays, as detailed below.

[0084] As is well known, drugs or other haptens can be derivatized to include a linking group having a chemical moiety that facilitates binding to a carrier or label. For linking groups for preparing haptens, see Mohammed Aslam and Alastair Dent, *Bioconjugation* (McMillan References, London, 1998).

[0085] Simply put, bromoacetamide can be prepared as follows: A appropriately substituted phenyl group having a protected amino group or carboxyl group is introduced to the 2,4-diamino-6-methyl-pteridinyl (DAMP) moiety to obtain a protected amine derivative having various linkers. After deprotection, this is acylated with an activated bromoacetamide derivative to obtain the bromoacetamide of the present invention.

[0086] The linking group may be a maleimide or vinyl sulfone that functions as a Michael acceptor for a thiol group acting as a nucleophile. The linking group of the MTX analog (DAMP-Y) may include a leaving group or a group that reacts with a nucleophile to form an adduct. A leaving group is a chemical moiety that is active when the MTX analog is conjugated with a label or carrier. As part of the conjugation process, one or more atoms of the leaving group are lost. Furthermore, conjugation with the label or carrier usually modifies the leaving group, and as a result, the linking group in the conjugate includes the modified residues. For convenience in this specification, the term “linking group” refers to both the linking group bonded to DAMP to form the MTX analog and the residues of the linking group after conjugation with the label or carrier.

[0087] In some exemplary embodiments described herein, DAMP-Y, or MTX analog, is derivatized with a carboxyl group-containing linking group (YZ) used to conjugate the analog to a label or carrier. In a typical conjugation step, the carboxyl group on the MTX analog is reacted with N-hydroxysuccinimide (NHS) to form an active ester. This active ester reacts with an amino group to form an MTX analog conjugate. The amino group can be present in low molecular weight molecules such as fluorescein or biotin derivatives, or in high molecular weight molecules (proteins) such as bovine serum albumin, keyhole linpuet cyanine, or peroxidase. In some cases, the MTX conjugate containing a carrier or label can be used as an immunogen or tracer, respectively.

[0088] In certain embodiments, the disclosure provides antibodies or antigen-binding fragments that bind to MTX analogs, such as DAMP derivatives conjugated with MTX and / or other molecules. In some embodiments, the antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO: 1, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to said sequence. In some embodiments, the antibody has the amino acid sequence of the light chain variable region of SEQ ID NO: 5, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to said sequence. In some embodiments, the antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO: 9, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to said sequence. In some embodiments, the antibody has the amino acid sequence of the light chain variable region of SEQ ID NO: 13, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to that sequence. In some embodiments, the antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO: 17, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to that sequence. In some embodiments, the antibody has the amino acid sequence of the light chain variable region of SEQ ID NO: 21, or an amino acid sequence having at least 80%, for example, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to that sequence.

[0089] In further embodiments, the antibody or its fragment may be a monoclonal antibody or a polyclonal antibody. Depending on the preparation method, in certain embodiments, the antibody of this disclosure may exist in a lyophilized state.

[0090] The antibodies used in this disclosure may include immunoglobulin molecules or portions thereof that can bind to a desired binding site. The immunoglobulin molecules of this disclosure may be essentially any class or isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Furthermore, structures known as nanobody or domain antibodies may also be used, and may include polypeptides containing one or more CDRs of an antibody known to bind to the corresponding binding site, provided that an effective amount of binding ability is retained.

[0091] In certain embodiments, the immunogens of this disclosure are MTX analogs containing a carrier. As used herein, the term “carrier” refers to a substance that is immunogenic in a selected host animal. Methods for preparing immunogens by ligating a hapten to a carrier are well known. The selection of carriers and routes of administration for inducing an immune response vary depending on the host animal.

[0092] Carriers are generally macromolecules, such as polymers. In some cases, carriers are macromolecular proteins derived from a different species than the host animal. Bovine serum albumin (BSA) and keyhole lymph hemocyanin (KLH) are frequently used as carriers for antibody induction in mice, rats, goats, rabbits, chickens, and sheep. Additional carriers, such as proteins used for conjugation with immunogens, are also widely known to those skilled in the art, and such embodiments are also included within the scope of this disclosure.

[0093] Typical preparations of immunogenic MTX analogs for inducing anti-MTX antibodies are described in the examples. In certain typical immunogenic preparations, RY is (CH2) n CO-NH-(carrier), where n is 1 to 12, and may contain one or more heteroatoms such as O, N, and S. In some cases, n is 1 to 8, and the carrier is BSA.

[0094] As used herein, the term “tracer” refers to a labeled analyte analog usable in competitive immunoassays. The tracers of this disclosure are MTX analogs in which a label is conjugated to MTX via a linking group.

[0095] The term "label" refers to a substance that can be detected directly or indirectly. Directly detectable labels include, for example, radionuclides and fluorochromes. Labels can also be detected indirectly through one or more reactions. Such labels include enzymes that are detected by producing a signal such as a colored product, chemiluminescence, fluorescence, or radioactive product. Such enzyme labels and their signal evolution systems are widely known to those skilled in the art. Other labels include those that utilize one of a specific binding pair, such as biotin / avidin. Other labels that can be used in immunoassay procedures include, and are well known, enzymes, radionuclides, fluorochromes, dioxetanes, acridinium esters, lanthanides and metal chelates, and biotin. In some cases, the label may be fluorochrome, acridinium ester, biotin, or enzymes such as HRP or G6PDH.

[0096] Suitable fluorochromes include xanthene dyes, such as fluorescein and rhodamine (e.g., tetramethylrhodamine isothiocyanate: TRITC), phycoerythrin (PE), allophycocyanin (APC), and Texas Red (Thermo Fisher, Waltham), with fluorescein being preferred. Although allophycocyanin and phycoerythrin are suitable fluorochromes, their molecular weight is too large for use in fluorescence-polarized immunoassays. Suitable fluoresceins include fluorescein isothiocyanate (FITC), (2-aminoethyl)-thioureidofluorescein (FTED), fluorescein-thiosemicarbazide (FTSC), (2-aminoethyl)-ureidofluorescein (FAMCO-E), erythrosine (tetraiodofluorescein), fluoresceinamine (FAM), and their derivatives (e.g., Oregon Green, Tokyo Green).

[0097] In certain embodiments, a tracer having a fluorescein residue linked to a linking group via the 5-position of the fluorescein moiety is referred to as isomer I. A tracer having a fluorescein residue linked to a linking group via the 6-position of fluorescein is referred to as isomer II. In tracers labeled with fluorescein and rhodamine, the lactone form is hardly present during fluorescence measurements, while the carboxylated form is mainly present as a salt. Fluorochrome may be of a homogeneous composition or a mixture of isomers. Furthermore, fluorochrome may be used as a lactone form or as a bioacceptable salt (e.g., Na, K, ammonium, and similar salts), thereby allowing it to exist in an ionized state in immunoassays.

[0098] Alternatively, carboxylic acids can be condensed with amines using other methods well known to those skilled in the art. Synthetic methods for forming carboxylic acid amides are well known and are described in M. Bodansky and A. Bodansky, *The Practice of Peptide Synthesis, 2nd Edition* (Springer-Verlag, New York, 1995). Methods for preparing immunogenic conjugates are also described in Cutris Williams, *Methods in Immunology and Immunochemistry* (Academic Press, 1977). These publications are incorporated herein by reference in their entirety. Furthermore, exemplary methods for preparing MTX analogs useful as tracers or immunogens are described in detail in the following Examples section. Examples of specific MTX analogs useful as immunogens, tracers, and especially enzyme conjugates are listed in Table 3 below.

[0099] Broadly speaking, these methods can be classified into those that do not use labeling (e.g., mass spectrometry) or those that use labeling, such as immunoassays using antibodies against analytes. Mass spectrometry exhibits high specificity when combined with chromatography such as GC-MS or LC-MS / MS. However, GC-MS and LC-MS / MS methods are time-consuming, require specialized equipment, highly trained analysts, and involve complicated sample preparation, and are expensive. Furthermore, these methods require excessively large sample volumes for use in pediatric testing, except in cases of abnormally high MTX concentrations. In short, mass spectrometry is not routinely used for typical clinical chemistry or hospital laboratory monitoring of MTX as a therapeutic drug. Immunosays for MTX have been developed based on heterogeneous formats requiring multiple washing steps, as well as simpler homogeneous formats that only require mixing and measurement.

[0100] More recently, a competitive immunoassay (US Patent No. 11,054,430) has been developed using antibodies that detect free MTX but do not cross-react with DAMPA, while still cross-reacting with isolated 7-OH MTX. These antibodies were used in the competitive immunoassay and were prepared against specific immunogens linked via N-10 (see Figure 1 in US Patent No. 11,054,430). As haptens, derivatives related to chemical modifications of the N-10 linked derivatives of MTX were used. Antibodies were used to detect MTX, and some were selected to minimize cross-reactivity with DAMPA, but still cross-react with 7-OH MTX. This method is heterogeneous, requiring washing, and has limited sensitivity due to impaired accuracy at the limit of quantification. Other cross-reactivity with folic acid, folinic acid, dihydrofolate, tetrahydrofolate, and trimethoprim exceeds 0.1%.

[0101] One embodiment of the present disclosure provides a compound represented by the following formula (1). TIFF2026514355000004.tif62169In formula, R 1 is -YZ, Y is a linking group, Z is selected from hydrogen, OH, SH, S-acyl, O-alkyl, halogen, NH2, epoxy, maleidyl, haloacetamide, carboxyl, activated carboxyl, alkyne, azide, immunogenic carrier, protein, and label.

[0102] Embodiments of this disclosure include antibodies. In some embodiments, the antibodies of this disclosure specifically bind to any compound of this disclosure, including the compound of formula (1) as described elsewhere in this specification.

[0103] Nucleic acids encoding any of the antibodies in this disclosure are also provided, as well as expression vectors containing such nucleic acids, and cells containing such nucleic acids and expression vectors.

[0104] The antibodies and enzymes disclosed herein can be used in various assays and methods for measuring, quantifying, and / or detecting the presence of MTX in a sample. In some embodiments, a competitive assay format is used. In certain such formats, the antibody or enzyme is immobilized on a solid support and used to capture labeled MTX and (if present in the sample) unlabeled MTX. If MTX is not present in the sample, the labeled MTX binds to the antibody / enzyme on the solid support and generates a signal. Free MTX in the sample competes with the labeled MTX for the binding site of the antibody / enzyme, resulting in a decrease in the signal. The signal is inversely proportional to the binding of unlabeled MTX in the sample.

[0105] The antibodies and enzymes described herein can be used in combination with a sample to perform assays. The sample may be a biological sample, such as a tissue extract, tissue, or liquid used in immunohistochemistry. The liquid sample may be derived from blood, plasma, serum, or buffer.

[0106] The antibodies and enzymes described herein may be linked or bound to various components or parts in order to perform assay functions. For example, in some embodiments, the antibodies and enzymes described herein may be bound to a solid support or carrier indirectly via covalent or noncovalent attachment to form capture molecules. In the case of indirect binding, intermediate linkers may be used to link the components. Suitable intermediate linkers include amino groups, carboxylate groups, thiols, biotin, ligands, and other chemical bonds. Suitable solid supports or carriers include glass surfaces (e.g., glass slides or beads), plastic surfaces, metal surfaces, polystyrene surfaces (e.g., beads or plates), nitrocellulose surfaces, microparticles, nanoparticle surfaces, plates, wells, and paramagnetic beads or magnetic beads coated with avidin or streptavidin, or having other surface functional groups that promote binding affinity.

[0107] In some embodiments, labeled MTX is used in a competitive assay format. In certain embodiments, a portion of the MTX may be linked or bonded to a label directly or indirectly by covalent or noncovalent bonding to form labeled MTX. In the case of indirect bonding, an intermediate linker may be used as described herein.

[0108] As described above, in some embodiments, an antibody or fragment thereof, or an aptamer, may be bound to a solid support to form a capture molecule in the assay or method of the present disclosure. This binding may occur before or after contacting the antibody or fragment thereof, or the aptamer, with a sample. The antibody or fragment thereof, or the aptamer, may be bound to the solid support directly (e.g., by covalent bond) or indirectly (e.g., using a binding partner).

[0109] Similarly, in some embodiments, MTX molecules may be bound to a label to form labeled MTX in the assay or method of the present disclosure. This binding may occur before or after contact of the MTX with the sample. MTX may be bound to the label directly (e.g., by covalent bonding) or indirectly (e.g., using a binding partner).

[0110] Examples of suitable binding partners include biotin / streptavidin, antibody / antigen, antibody / Fc receptor, antibody from a first animal and antibody from a second animal against the first antibody, Fc / Fc receptor, and 6-His / Ni 2+ Examples include, but are not limited to, 6-His / cobalt and 6-His / divalent cationic resins.

[0111] In other embodiments, the binding pair may be streptavidin and biotin, or two antibodies that bind to each other, for example, an antibody that binds to the Fc portion of another antibody. In yet another embodiment, binding may occur via interactions between multiple binding pairs. Substantially any method can be used for binding the antibody to the solid support or for binding the MTX to the label (e.g., direct or indirect binding). In some embodiments, the antibody may contain biotin and the solid support may contain streptavidin, and vice versa.

[0112] In some embodiments, the label may be any label corresponding to a suitable detection method. Suitable detection modes include, but are not limited to, absorbance, fluorescence or emission using the label or compound, chemiluminescent compounds, enzyme labels, fluorophores, colorimetric compounds, radioactive labels, catalysts, colorimetric compounds or labels, labeled antibodies, latex particles, magnetic particles, radioactive elements, fluorescent dyes, phosphorescent dyes, lanthanides, gold particles, silver colloid particles, selenium colloid particles, metal chelates, ferrocenes, coenzymes, electroactive groups, oligonucleotides, or stable radicals. Metal chelates may be ruthenium chelates for electrochemiluminescence, or osmium metal chelates or lanthanide chelates (e.g., europium, samarium, or terbium chelates) for time-resolved fluorescence. Detection methods include, but are not limited to, any known detection methods, including, colorimetric, radioisotope, fluorescence, immunofluorescence, luminescence, bioluminescence, electrochemiluminescence (ECL), amperometry using current or impedance, surface-enhanced Raman scattering (SERS), and surface plasmon resonance (SPR).

[0113] In some embodiments, the detection method may be absorbance read in endpoint mode or kinetic mode. A colorimetric compound (colorimetric compound) functions as a label and can be detected or quantified on a reaction chamber such as a cuvette or flow cell, or on a disposable container or lateral flow strip. A solid support may function to hold the label-bound antibody near the electrode in the electrochemical reading reaction chamber during detection. The solid support may be a lateral flow device or a surface plasmon generating surface.

[0114] In some embodiments, the solid carrier and / or label may be derived from a lyophilized composition that is rehydrated with the sample when used in the assay. The lyophilized composition may contain standards and / or other necessary assay-specific components, such as buffers, reagents, surfactants, preservatives, salts, proteins, antibodies, etc. The solid carrier and label may be lyophilized as separate compositions and then rehydrated with the sample. Alternatively, the solid carrier and label may be lyophilized in the same composition and then rehydrated with the sample.

[0115] The antibodies and enzymes of this disclosure can be used in heterogeneous immunoassays such as enzyme-linked immunosorbent assays (ELISA) and electrochemiluminescence assays (ECL) for detecting the presence of MTX, or in homogeneous immunoassays that do not require a washing step to separate the bound and unbound parts, such as enzyme immunoassay (EMIT), enzyme competitive immunoassay (CEDIA), chemiluminescence immunoassay (LOCI), and various assay formats including turbidimetric formats such as turbidimetric immunoassay (QMS) (Thermo Fisher), particle-enhanced turbidimetric immunoassay (PETIA) (Abbott), and kinetic immunoassays (KIMs) (Roche). In one embodiment of the present disclosure, an assay method for detecting and / or quantifying MTX in a sample may include the steps of: mixing the sample with a capture molecule and labeled MTX in a solution (the capture molecule being able to bind to the labeled MTX, and the MTX present in the sample competing with the capture molecule for binding to the labeled MTX); and detecting the amount of labeled MTX bound to the capture molecule via a signal generated by a label on the labeled MTX bound to the capture molecule (the signal being inversely proportional to the amount of MTX present in the sample).

[0116] In some embodiments, the capture molecule comprises an antibody or a fragment thereof conjugated to a solid carrier. In some embodiments, the capture molecule may be an antibody or a fragment thereof conjugated to a solid carrier, and the antibody or a fragment thereof conjugates to MTX and / or other molecules. For example, the antibody may selectively conjugate to a molecule conjugated with MTX compared to the same molecule not conjugated with MTX. When it is stated that an antibody conjugates to MTX, it is understood that the antibody comprises an antibody that selectively conjugates to a molecule conjugated with MTX compared to the same molecule not conjugated with MTX.

[0117] In some embodiments, labeled MTX includes MTX molecules or other molecules conjugated with MTX covalently linked to the label. In some embodiments, labeled MTX includes an MTX conjugate covalently linked to the label.

[0118] In some embodiments, the conjugate of DAMP, which is an MTX analog, is a DAMP derivative modified at the phenyl ring of formula (1). The modification of the phenyl ring can be carried out via alkyl, carbonyl, or heteroatoms such as O, N, and S. In some embodiments, the conjugate of DAMP, which is an MTX analog, is an MTX derivative in which the gamma (γ) position of the carboxyl group of MTX is chemically or naturally modified.

[0119] The steps in the method of this disclosure do not have to be performed in the order described herein and may be performed in a different order. Furthermore, the sample may be incubated for a certain period of time before the washing step and the removal of unbound or excess substances. Additionally, additional washing steps for the removal of substances during the assay may be performed at other times in the method, for example, after the addition of each assay capture molecule and / or before the detection step.

[0120] In other embodiments, the capture molecule and the sample are combined before the addition of the labeled MTX. For example, the solution containing the sample and the capture molecule may be incubated for a certain period of time before the addition of the labeled MTX.

[0121] The components or reagents used in the embodiments of the assays disclosed herein can be lyophilized using standard lyophilization methods. For example, a solution containing the desired components, such as labeled MTX or a capture molecule, can be prepared, the solution can be dropped into a freezing medium (e.g., liquid nitrogen) to form frozen spheres, and then the frozen spheres or pellets can be lyophilized.

[0122] In some embodiments of the assay, a lyophilized composition containing a capture molecule, labeled MTX, or both is rehydrated with the sample. This embodiment is advantageous because the sample is not essentially diluted during the assay, and higher sensitivity may be obtained because more MTX is present in the undiluted sample compared to the same volume of diluted sample. In some embodiments, the sample is diluted before being combined with other reagents. In some embodiments, the sample is not diluted before being combined with other reagents.

[0123] This disclosure also provides a method for producing an antibody. The method includes culturing the cells of this disclosure under conditions suitable for the cells to express the antibody, thereby producing the antibody.

[0124] Aspects of the present disclosure further include compositions. Compositions of the present disclosure may include any of the antibodies, nucleic acids, expression vectors, and / or cells of the present disclosure.

[0125] The Disclosure also provides a method for determining the amount of at least one MTX analyte in a medium. In certain embodiments, such a method includes combining a sample presumed to contain at least one MTX analyte with an antibody of the Disclosure in a medium. Such a method further includes determining the presence or absence of a complex containing the MTX analyte and the antibody, the presence of which indicates the presence of the MTX analyte in the sample.

[0126] Aspects of the present disclosure further include kits. According to some embodiments, such kits are used to determine the amount of at least one MTX analyte in a sample. In certain embodiments, a kit of the present disclosure includes an antibody of any of the present disclosure and instructions for determining the amount of at least one MTX analyte in a sample using such antibody. Such a kit may further include any compound of formula (1) of the present disclosure. According to some embodiments, a kit of the present disclosure includes a compound of formula (1) of the present disclosure and instructions for determining the amount of at least one MTX analyte in a sample using such compound. Such a kit may further include any antibody of the present disclosure.

[0127] Compounds, conjugates, and their synthesis Homogeneous enzyme immunoassays rely on the use of enzyme-SBP member conjugates whose enzyme activity can be strongly modulated by binding to an SBP partner. This disclosure provides enzyme-SBP member conjugates and antibodies for performing assays useful for homogeneous immunoassays.

[0128] In certain embodiments, protein immunogens are synthesized and used to prepare antibodies specific to compounds such as MTX analytes. These antibodies may be used in methods for detecting MTX analytes in samples presumed to contain them. Labeled conjugates may be prepared and used in the above methods. As described above, effective quantification of the presence of MTX analytes in a sample can be achieved.

[0129] Immunogens and labeled conjugates may contain MTX analogs linked to proteins or labels via phenyl groups. In some cases, these conjugates may be referred to as protein conjugates or labeled conjugates, respectively.

[0130] The compounds of this disclosure include compounds useful for producing antibodies relating to this disclosure. Furthermore, the compounds of this disclosure include conjugates useful for immunoassays described herein. In certain embodiments, the compounds include compounds represented by the following formula (1). TIFF2026514355000005.tif59168 formula, R 1 is -YZ, Y is a linking group, Z is selected from the group consisting of hydrogen, OH, SH, S-acyl, O-alkyl, O-sulfonate, halogen, NH2, epoxy, maleidyl, haloacetamide, carboxyl, activated carboxyl, azide, alkene, immunogenic carrier, protein and label, and salts thereof.

[0131] In some embodiments, Z is a protein. For example, the protein may be an immunogenic carrier. The immunogenic carrier is conjugated to an MTX hapten, thereby enabling the production of an antibody that can specifically bind to the hapten. For example, the immunogenic carrier may be selected from hemocyanin, globulin, albumin, and polysaccharides. In some cases, the immunogenic carrier may be bovine serum albumin (BSA). In some cases, the immunogenic carrier may be keyhole limpet hemocyanin (KLH). In certain embodiments, the immunogenic carrier may be modified to include one or more functional groups. The functional groups on the modified immunogenic carrier may be reactive functional groups that facilitate the binding of the immunogenic carrier to the linking group in the compound of formula (1).

[0132] In some embodiments, the 2,4-diamino-6-aminomethyl-pteridinylphenyl (DAMP) hapten is linked via an alkyl, ether, thioether, azo, keto, or alkene functional group. Therefore, in some cases, the linking group includes an alkyl group or substituted alkyl group bonded to the phenyl group of the pteridinylmethylamino moiety.

[0133] In certain embodiments, such haptens are used to produce antibodies specific to MTX.

[0134] In certain embodiments, Z is a label. A label is a molecule that generates or can be induced to generate a detectable signal. For example, the label can be an enzyme, and examples of enzymes include those selected from alkaline phosphatase, β-galactosidase, and horseradish peroxidase. In some embodiments, the label is an enzyme, and the enzyme is glucose-6-phosphate dehydrogenase (G6PDH). In some cases, G6PDH is a mutant G6PDH that contains one or more amino acid residue substitutions compared to the wild type. For example, the mutant G6PDH can contain cysteine substitutions in each subunit. In some cases, the linking group can be attached to the cysteine residue of the G6PDH enzyme. In certain embodiments, the label can be modified to include one or more functional groups. The functional groups on the modified label can be reactive functional groups that facilitate attaching the label to the linking group in the compound of formula (1).

[0135] In some embodiments, the MTX analog is derived from an acylated phenyl ring that is ortho, meta, or para to MTX (see Figure 2). Thus, in some cases, the linking group contains an acyl group or a substituted acyl group attached to the phenyl ring of the 2,4-diamino-6-aminomethyl-pteridinylphenyl (DAMP) moiety (i.e., the R 1 -phenyl ring). In certain embodiments, such derivatives are used to create conjugates useful in the immunoassays described herein.

[0136] In certain embodiments, Z is a protein. The protein can be any suitable protein, which contains amino acid residues such as dipeptides and tripeptides, and the number of such residues can be any number, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. In certain embodiments, the protein can be modified to contain one or more functional groups. The functional groups on the modified protein can be reactive functional groups that facilitate the linking of the protein to the linking group in the compound of formula (2). In some cases, the protein is acylated. In some cases, the protein is alkylated.

[0137] The linking group may contain approximately 1 to 25 atoms (excluding hydrogen atoms) and may contain a chain of 2 to 15 atoms (excluding hydrogen atoms), each atom independently selected from carbon, oxygen, sulfur, nitrogen, halogen, and phosphorus. In some embodiments, the linking group contains 1 to 15 carbon atoms and / or 0 to 6 heteroatoms. Examples of linking groups include, but are not limited to, -(CH2) n C(O)-, -C(O)(CH2) n -, -C(O)(CH2) n NHC(O)-, -C(O)(CH2) n NHC(O)(CH2) n -,-(CH2) n SCH2C(O)-, -(CH2) n C(O)NH(CH2) n -,-(CH2) n NHC(O)-,-(CH2) n NHC(O)(CH2) n -,-(CH2) n NHC(O)(CH2) n O(CH2) n NHC(O)(CH2) n -,-(CH2) n NHC(O)(CH2) n NHC(O)(CH2) n -, -NH(CH2) n C(O)-, -(CH2) n -, -C(O)NH(CH2CH2O) m (CH2)n NHC(O)(CH2) n -, and -(CH2) n (Heterocykrill) S(CH2) n Examples include C(O)- and its salts, where each m is an independent integer between 1 and 10, and each n is an independent integer between 1 and 10. In certain embodiments, the linking group is -C(O)NH(CH2CH2O)2(CH2)2NHC(O)CH2-. In certain embodiments, the linking group is -CH2NHC(O)(CH2)7NHC(O)CH2-. In certain embodiments, the linking group is -CH2NHC(O)(CH2)2O(CH2)4NHC(O)CH2-. In certain embodiments, the linking group is -CH2NHC(O)(CH2)2NHC(O)CH2-. In certain embodiments, the linking group is -CH2NHC(O)CH2-. In certain embodiments, the linking group is -C(O)(CH2) n NHC(O)(CH2) n - and for example, -C(O)(CH2CH2)NHC(O)(CH2)-. In certain embodiments, the linking group is -(CH2) n (Heterocykrill) S(CH2) n It is C(O)-, for example -(CH2CH2CH2CH2)(2,5-dioxopyrrolidine-1-yl)S(CH2)C(O)-.

[0138] The number of heteroatoms in the linking group is 0 to 6, for example, about 1 to 5, 2 to 5, or 3 to 5. The linker can be aliphatic or aromatic. When heteroatoms are present, oxygen may exist as oxo or oxy and may bond to carbon, sulfur, nitrogen, or phosphorus; nitrogen may exist as nitro, nitroso, or amino and may bond to carbon, oxygen, sulfur, or phosphorus; sulfur may be similar to oxygen; phosphorus may bond to carbon, sulfur, oxygen, or nitrogen, for example, phosphonates and monoesters or diesters of phosphoric acid. Common functional groups that form covalent bonds between the linking group and the molecule to be conjugated include alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azos, thioethers and carboxylates, sulfonates, and phosphoric acid esters, amides and thioesters.

[0139] In certain embodiments, when the linking group has a non-oxocarbonyl group (including nitrogen and sulfur analogs), a phosphate group, an amino group, an alkylating agent such as a haloalkyl or tosylalkyl, an oxy (a mercapto which is a hydroxyl or sulfur analog), an oxocarbonyl (e.g., an aldehyde or ketone), or an active olefin such as a vinyl sulfone or an α- or β-unsaturated ester, these functional groups can be linked to an amino group, a carboxyl group, an active olefin, or an alkylating agent (e.g., bromoacetyl). When an amino is linked to a carboxylic acid or its nitrogen derivative or a phosphate, amides, amidines, and phosphoramides can be formed. When a mercaptan is linked to an active olefin, a thioether can be formed. A thioether can also be formed when a mercaptan is linked to an alkylating agent. When an aldehyde is linked to an amine under reducing conditions, an alkylamine can be formed. When a carboxylic acid or phosphate is linked to an alcohol, an ester can be formed. For information on various linking groups, see, for example, Cautrecasas, *J. Biol. Chem.* (1970), 245:3059.

[0140] To develop assays against MTX, the chemical structure of MTX that retains the 2,4-diaminopteridinyl-methylaminomethyl-phenyl (DAMP) moiety is used. For example, MTX has multiple nitrogen atoms as part of the pteridine nucleus and also has an aminomethyl group (N-10 as described in U.S. Patent No. 10,054,430) (see formula (1) for numbering). Alkyl and acyl linked derivatives derived from the phenyl moiety retain the basic structure (core) of the diaminopteridinyl-methylaminomethyl (DAMP) structure. Specific binding via the phenyl group generates a chemical structure that maintains high complementarity with MTX for preparing immunogens and, accordingly, producing antibodies.

[0141] This disclosure provides the design of MTX haptens and immunogens by modifying the glutamic acid moiety of MTX. The haptens derived from the MTX analogs DAMP-1, 2, 3, 4, 5, and 6 of this disclosure and their synthesis are shown in Figures 4-15. By introducing a linking group to the phenyl ring, antibodies that can react specifically with MTX analytes can be provided because the analytes share a pteridinylaminomethylaminophenyl group. Thus, this disclosure provides MTX analogs and immunogens useful for the various immunoassays described herein.

[0142] Compounds useful for producing antibodies and conjugates relating to this disclosure can be synthesized according to the general synthetic methods described below. The compound of formula (1) can be prepared by standard methods. The following reaction schemes are merely examples of these methods and do not limit this disclosure.

[0143] A) Haptens The introduction and removal of protecting groups such as tert-butoxycarbonyl (Boc) are typically performed to protect the amino group in preparation for subsequent acylation with activated haloacetic acid derivatives. Appropriate protecting groups are described in detail in patent and technical literature. For example, see M. Bodanszky, *Principles of Peptide Synthesis* (Springer Verlag, Berlin, Heidelberg, New York, Tokyo, 1984). Examples of such protecting groups include, but are not limited to, t-butoxycarbonyl (t-Boc), fluorenylmethyloxycarbonyl (Fmoc), acetamidemethyl (Acm), triphenylmethyl (Trt), benzyloxycarbonyl, biphenylisopropyloxycarbonyl, 1-amyloxycarbonyl, isobornyloxycarbonyl, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-1,1-dimethylethoxycarbonyl, bromobenzyloxy, carbamyl, and formyl. The specific protecting group selected depends on the nature of the reaction to be carried out and the reaction conditions such as temperature and pH.

[0144] B) Immunogen Functionalized haptens (e.g., DAMP1-6, MTX, DAMPA) can be conjugated to proteins. Haptens with carboxyl groups can be directly conjugated to the ε-amino group of lysine in proteins. Haloacetamides can usually be conjugated to thiols in a two-step procedure. Specifically, the lysine residue of the protein is activated by acylation of the ε-nitrogen with N-succinimidyl-S-acetylthioacetate (SATA), and then the S-acetyl group is hydrolyzed with hydroxylamine to generate a nucleophilic sulfhydryl group. The conjugation of the sulfhydryl-activated protein with the bromoacetamide-derivatized hapten forms a thioether-linked conjugate via a nucleophilic substitution reaction of the bromide. Suitable proteins (immunogenic carriers) include, but are not limited to, keyhole lymph hemocyanin, bovine thyroglobulin, and ovalbumin.

[0145] The MTX analog contains a bromoacetamide functional group for modifying the thiol group of a thiol-containing protein. The synthesis of the MTX immunogen DAMP-YS-KLH, which has a linking group (L) on the phenyl ring of the MTX analog DAMP, begins with the synthesis of DAMP-Y shown in Figures 4-10, and its preparation is described in Examples 1-6. The reaction of the amino group derived from keyhole lymptohemocyanin (KLH) with N-succinimidyl-S-acetylthioacetate generates a protected sulfhydryl, which is deprotected with hydroxylamine to become reactable with the DAMP-bromoacetamide of the present invention. The reaction of the thiol-modified KLH-SH with DAMP-bromoacetamide is carried out in sodium phosphate buffer (0.1 M, pH 8.0) to produce the desired immunogen DAMP-YS-KLH shown in Figure 11. Immunogen DAMP-YS-KLH can be purified by chromatography using a Sephadex G-25 column with buffer. The concentration of immunogen DAMP-YS-KLH can be measured using a protein assay such as the Pierce® Rapid Gold BCA protein assay kit. Immunogen DAMP-YS-KLH can be used to immunize rabbits for antibody production.

[0146] C) Enzyme Conjugate The DAMP-L hapten of the present invention has a bromoacetamide functional group and can be used in reactions with proteins containing thiol groups. The conjugation of DAMP-Y bromoacetamide to G6PDH containing cysteine ​​is shown in Figure 10, and its preparation is described in Example 15.

[0147] Haptens derived from DAMP-Y can be used for immunogen preparation. Haptens DAMP-Y(1-6) can be used for G6PDH conjugate preparation. Immunogen DAMP-LS-KLH can be used to induce antibody production. In certain embodiments, the produced antibodies can show good modulation to MTX analytes in enzyme-based assay formats. Since antibodies can be successfully produced using immunogen DAMP-YS-KLH in several embodiments, it is suggested that such antibodies may be useful in enzyme-based MTX immunoassays described later. Antibodies and their preparation Aspects of this disclosure include antibodies that specifically bind to MTX. In some embodiments, the antibodies of this disclosure specifically bind to any of the compounds of this disclosure, for example, any of the compounds of general formula (1) described elsewhere.

[0148] The term “antibody” (sometimes used synonymously with “immunoglobulin”) encompasses polyclonal antibodies (e.g., rabbit polyclonal antibodies) and monoclonal antibody preparations, which may include antibodies or immunoglobulins of any isotype (e.g., IgG (IgG1, IgG2, IgG3, or IgG4, etc.), IgE, IgD, IgA, IgM, etc.); full-length antibodies consisting of tetramers (further composed of dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); antibody fragments retaining specific compound-binding ability (e.g., including full-length antibodies or single-chain antibody fragments, and not limited to single-chain Fv(scFv), Fab, (Fab')2, (scFv')2, diabody, etc.); chimeric antibodies; monoclonal antibodies; human antibodies; and fusion proteins comprising an antigen-binding site of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from IgG, Fv, single-chain antibodies, scFv, Fab, F(ab')2, or Fab'. The antibody may be further conjugated to other parts, such as members of a specific binding pair (e.g., biotin (a member of the biotin-avidin specific binding pair)).

[0149] Immunoglobulin polypeptides include κ (kappa) and λ (lambda) light chains, as well as α (alpha), γ (gamma) (IgG1, IgG2, IgG3, IgG4), δ (delta), ε (epsilon), and μ (mu) heavy chains, or equivalents in other types. The "light chain" of full-length immunoglobulins (typically about 25 kDa or about 214 amino acids) contains a variable region of about 110 amino acids at the NH2 terminus and a κ (kappa) or λ (lambda) constant region at the COOH terminus. Similarly, the "heavy chain" of full-length immunoglobulins (about 150 kDa or about 446 amino acids) contains a variable region (about 116 amino acids) and the aforementioned heavy chain constant region (e.g., γ (gamma) (about 330 amino acids)).

[0150] The variable regions of the light or heavy chains of immunoglobulins consist of a "framework" region (FR), which is interrupted by three highly variable regions (also called "complementarity-determining regions" or "CDRs"). The extents of the framework region and CDRs have already been defined; see, for example, E. Kabat et al., *Sequences of Proteins of Immunological Interest* (US Department of Health and Human Services, 1991) and Lefranc et al., *IMGT, the international ImMunoGeneTics information system®* (Nucl. Acids Res., 2005, Vol. 33, D593-D597). Details of the IMGT system (including its development history and comparisons with other systems) are available on the World Wide Web at imgt.cines.fr / textes / IMGT ScientificChart / Numbering / IMGTnumberingsTable.html. Within a species, the sequences of the framework regions of different light or heavy chains are relatively conserved. The antibody framework region, i.e., the sum of the framework regions of the constituent light and heavy chains, plays a role in positioning and aligning the CDR. The CDR is primarily responsible for binding to the antigen epitope. All CDRs and frameworks provided herein are defined based on the aforementioned IMGT unless otherwise specified.

[0151] Thus, an "antibody" encompasses a protein having one or more polypeptides, which can be genetically encoded, for example, by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include numerous immunoglobulin variable region genes, in addition to the constant region genes of κ (kappa), λ (lambda), α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mu). The light chain is classified as either κ (kappa) or λ (lambda). The heavy chain is classified as γ (gamma), μ (mu), α (alpha), δ (delta), or ε (epsilon), which define the classes of immunoglobulins, namely IgG, IgM, IgA, IgD, and IgE, respectively.

[0152] Typical immunoglobulin (antibody) structural units are known to include tetramers. Each tetramer consists of two identical polypeptide chain pairs, each pair having one "light chain" (approximately 25 kD) and one "heavy chain" (approximately 50-70 kD). The N-terminus of each chain defines a variable region consisting of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. (Light chain variable region (V)) L ) and "Heavy chain variable region (V H The terms ) refer to these light and heavy chains, respectively.

[0153] Antibodies contain not only complete immunoglobulins but also numerous well-characterized fragments that can be genetically encoded or produced by digestion by various peptidases. For example, pepsin digests antibodies below the disulfide bond in the hinge region, and F(ab')2, i.e., the light chain, is the heavy chain variable region (V HThis generates a Fab dimer linked to )-CH1 by a disulfide bond. F(ab')2 is reduced under mild conditions, and the disulfide bond in the hinge region is cleaved, converting the (Fab')2 dimer to a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, edited by WEPaul (Raven Press, NY, 1993)). Various antibody fragments are defined based on the digestion of a complete antibody, but those skilled in the art will understand that such Fab' fragments can be synthesized chemically or de novo using recombinant DNA techniques. Thus, the term “antibody” as used herein includes antibody fragments produced by modification of full-length antibodies, or antibody fragments synthesized de novo using recombinant DNA techniques, including, but not limited to, Fab'2, IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies. In certain embodiments, the antibodies of this disclosure are selected from IgG, Fv, single-chain antibodies, scFv, Fab, F(ab')2, and Fab'.

[0154] The terms "specifically bind," "specific," "immunoreactivity," "immune response capacity," and "antigen-binding specificity," when used in reference to antibodies, refer to a binding reaction that binds highly selectively to an antigen or its fragments, and can determine the presence of that antigen even in the presence of heterogeneous antigens. Therefore, under given immunoassay conditions, a particular antibody will bind to a specific antigen and will not bind to other antigens present in the sample in significant amounts. In order to specifically bind to an antigen under such conditions, it may be necessary for the antibody to be selected to have specificity for a particular antigen. For example, the antibody may bind specifically to a compound and not show the same degree of binding to other molecules present in the sample.

[0155] In some embodiments, the antibody of this disclosure is, for example, about 105 M -1 An antibody is said to "specifically bind" to a compound if it has an affinity or dissociation constant Ka (i.e., the equilibrium association constant in a particular binding reaction, in units of 1 / M) of the above. In a particular embodiment, the antibody binds to the compound with a Ka of approximately 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 The above conditions are met for bonding. A "high affinity" bond is defined as a Ka value of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 This refers to the above-mentioned bonds. Alternatively, affinity is the equilibrium dissociation constant (K) in a particular bonding reaction. D It can also be defined as ), and its unit is M, for example 10 -5 M~10 -13 M is less than or equal to M. In some embodiments, specific binding means that the antibody binds to the compound, K D about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12This means binding at M or less. The binding affinity of an antibody to a compound can be easily determined using conventional techniques such as competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., using a BIAcore 2000 instrument and following the manufacturer's general procedures), bioluminescence interferometry (BLI, ForteBio), and radioimmunoassay.

[0156] Whether the first antibody "competes" with the second antibody for binding to the compound can be easily determined by using a competitive binding assay known to those skilled in the art. Competitive antibodies can be identified, for example, by an antibody competition assay. For example, a sample of the first antibody is bound to a solid support. Next, a sample of the second antibody, which is presumed to compete with the first antibody, is added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to different, independent sites on the compound, the labeled antibody will bind to a similar extent regardless of the presence or absence of a presumed competing antibody (unlabeled antibody). However, if the interaction sites are the same or overlap, the unlabeled antibody will compete, reducing the amount of labeled antibody that binds to the compound. If there is an excess of unlabeled antibody, little or no binding of the labeled antibody will occur.

[0157] As used herein, a "competitive antibody" refers to an antibody that reduces the binding of an antibody to the compound by about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. Details of procedures for performing such competitive assays are well known to those skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pages 567-569, ISBN: 0-87969-314-2). Such assays can be quantified by using purified antibodies. A standard curve can be created by using the same antibody for both the labeled antibody and the competitive antibody, i.e., titrating against itself. The ability of the unlabeled competitive antibody to inhibit the binding of the labeled antibody on the plate can be evaluated by titration. The results can be plotted and the concentrations required to achieve the desired level of binding inhibition can be compared.

[0158] According to some embodiments, the antibodies of the present disclosure a V containing the amino acid sequence RSDHWIC (SEQ ID NO: 2) H CDR1, a V containing the amino acid sequence CIYIGSGTFVRSGTTYYASWAKG (SEQ ID NO: 3) H CDR2, and a V containing the amino acid sequence GFYATDGYGGPSYLNL (SEQ ID NO: 4) H a heavy chain variable region (V H ) polypeptide containing CDR3, and a V containing the amino acid sequence QASESISSYCS (SEQ ID NO: 6) L CDR1, a V containing the amino acid sequence RASTLES (SEQ ID NO: 7) L CDR2, and a V containing the amino acid sequence QSYAYSSPDSYGST (SEQ ID NO: 8) L a light chain variable region (V L ) polypeptide containing CDR3, and Competitive with respect to the binding of an antibody to a compound of formula (1).

[0159] In certain embodiments, the antibody comprises six CDRs set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. According to some embodiments, the antibody comprises a heavy chain variable region (V H ) polypeptide having an amino acid sequence with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, and a light chain variable region (V L ) polypeptide having an amino acid sequence with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0160] In some cases, the antibody comprises three heavy chain CDRs set forth in SEQ ID NOs: 2-4 and has amino acid substitutions in the sequence region of SEQ ID NO: 1 other than the CDRs, and the resulting heavy chain variable region (V H ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Similarly, in some cases, the antibody comprises three light chain CDRs set forth in SEQ ID NOs: 6-8 and has amino acid substitutions in the sequence region of SEQ ID NO: 5 other than the CDRs, and the resulting light chain variable region (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. According to some embodiments, the antibodies of the present disclosure comprise a V H CDR1 comprising the amino acid sequence SSDHWIC (SEQ ID NO: 10), a V H CDR2 comprising the amino acid sequence CIYIGSGTFVSSGTTYFASWAKG (SEQ ID NO: 11), and a V comprising the amino acid sequence GFYYTDGSGGPSYLNL (SEQ ID NO: 12)H A heavy chain variable region (V H ) polypeptide containing a CDR3, A V containing the amino acid sequence QASQTIYSYLS (SEQ ID NO: 14) L CDR1, A V containing the amino acid sequence SASTLAS (SEQ ID NO: 15) L CDR2, and A V containing the amino acid sequence QSYMYSSSSFGST (SEQ ID NO: 16) L A light chain variable region (V L ) polypeptide containing a CDR3, Competitive with respect to the binding of the antibody to the compound of formula (1).

[0161] In certain embodiments, the antibody comprises the six CDRs set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. According to some embodiments, the antibody comprises a heavy chain variable region (V H ) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region (V L ) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 13.

[0162] In some cases, the antibody comprises the three heavy chain CDRs set forth in SEQ ID NOs: 10-12 and has amino acid substitutions in the sequence region of SEQ ID NO: 9 other than the CDRs, and the resulting heavy chain variable region (V H ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. Similarly, in some cases, the antibody comprises the three light chain CDRs set forth in SEQ ID NOs: 14-16 and has amino acid substitutions in the sequence region of SEQ ID NO: 13 other than the CDRs, and the resulting light chain variable region (V LThe polypeptide contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 13.

[0163] According to some embodiments, the antibodies of this disclosure are V containing the amino acid sequence KYYMT (SEQ ID NO: 18) H CDR1, V containing the amino acid sequence VTWSGGMTYYASWAKG (SEQ ID NO: 19) H CDR2, and V containing the amino acid sequence ERDYFDGYIGNDI (SEQ ID NO: 20) H The heavy chain variable region (V) including CDR3 H ) polypeptides and, V containing the amino acid sequence QSSQSVWSRHLS (SEQ ID NO: 22) L CDR1, V containing the amino acid sequence KASTLAS (SEQ ID NO: 23) L CDR2, and V containing the amino acid sequence LGGYTCIRDDCRA (SEQ ID NO: 24) L Light chain variable region including CDR3 (V L ) polypeptides and, The antibody and the compound of formula (1) compete for binding.

[0164] In certain embodiments, the antibody comprises six CDRs described in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. According to some embodiments, the antibody comprises a heavy chain variable region (V) containing an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with the amino acid sequence described in SEQ ID NO: 17. H ) polypeptide and a light chain variable region (V) containing an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more identity with the amino acid sequence described in SEQ ID NO: 21. L ) Includes polypeptides and

[0165] In some cases, the antibody contains three heavy chain CDRs as described in SEQ ID NOs. 18-20, and has amino acid substitutions in the sequence region of SEQ ID NO. 17 other than the CDRs, thereby obtaining a heavy chain variable region (V H The polypeptide contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 17. Similarly, in some cases, the antibody contains the three light chain CDRs described in SEQ ID NOs: 22-24 and has amino acid substitutions in the sequence region of SEQ ID NO: 21 other than the CDRs, thereby obtaining a light chain variable region (V L The polypeptide contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 21.

[0166] The above heavy chain variable region (V H ) polypeptide, light chain variable region (V L The amino acid sequences of the polypeptide and CDR are shown in Table 1 below.

[0167] [Table 1a]

[0168] [Table 1b]

[0169] [Table 1c]

[0170] [Table 1d] In certain embodiments, the antibodies of the present disclosure further specifically bind to analogs of MTX. Target analogs include, but are not limited to, pralatrexate and fotatrexate. According to some embodiments, the antibodies of the present disclosure have a reactivity greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45% or greater than 50% with respect to pralatrexate, based on the reactivity with MTX.

[0171] Aspects of the present disclosure further include nucleic acids. In certain embodiments, the nucleic acids of the present disclosure encode a heavy chain variable region (V H ) polypeptide, a light chain variable region (V L ) polypeptide, or both, which include V<000012,6>and / or V L containing the CDRs of any of the antibodies listed in Table 1, but are not limited thereto. Examples of nucleic acids having nucleotide sequences encoding exemplary antibodies of the present disclosure are shown in Table 2 below.

[0172]

Table 2a

[0173]

Table 2b

[0174]

Table 2c

[0175]

Table 2d

[0176]

Table 2e

[0177] [Table 2f]

[0178] This disclosure further provides expression vectors comprising any of the nucleic acids of this disclosure. These expression vectors, for example, express the heavy chain variable region (V) of the antibody of this disclosure in host cells. H ) and / or light chain variable region (V L ) can be used to express the V of the antibody of this disclosure. H and / or V L The expression of natural or synthetic nucleic acids encoding V is typically, H and / or V L This is achieved by operably linking the nucleic acid encoding the V to a promoter (constitutive or inducible) and incorporating the construct into an expression vector. These vectors may be capable of replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors include the transcription and translation termination sequences, start sequences, and V H and / or V L These vectors contain promoters useful for regulating the expression of nucleic acids encoding the nucleic acids. These vectors may optionally include a universal expression cassette containing at least one independent termination sequence, a sequence enabling cassette replication in both eukaryotes and prokaryotes (i.e., a shuttle vector), and a selection marker for both prokaryotes and eukaryotes. See Sambrook et al (1989). To obtain high levels of clonal nucleic acid expression, it is common to construct an expression plasmid containing a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation termination sequence.

[0179] Accordingly, aspects of the present disclosure further include cells, such as recombinant host cells. In certain embodiments, cells comprising any nucleic acid and / or expression vector of the present disclosure are provided. According to some embodiments, the heavy chain variable region (V) of the antibody of the present disclosure is provided. H) The first nucleic acid encoding the polypeptide and the light chain variable region (V) of the antibody. L Cells comprising a second nucleic acid encoding a polypeptide are provided. In certain embodiments, cells comprising a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid are provided. Cells of the Disclosure may be produced by introducing one or more nucleic acids and / or expression vectors of the Disclosure into host cells by methods known in the art, such as electroporation, lipofection, microinjection, etc.

[0180] The Disclosure also provides a method for producing the antibodies of the Disclosure. In certain embodiments, such a method comprises culturing the cells of the Disclosure (e.g., recombinant host cells) under conditions suitable for the cells to express antibodies, thereby causing the cells to produce antibodies. Suitable conditions for culturing cells to express antibodies can vary considerably. These conditions may include culturing the cells in a suitable container (e.g., a cell culture plate or its wells) in a suitable medium (e.g., cell culture media such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, etc.) at a suitable temperature (e.g., 32°C to 42°C, preferably 37°C) and pH (e.g., pH 7.0 to 7.7, preferably pH 7.4), and further in an environment having a suitable carbon dioxide (CO2) concentration (e.g., 3% to 10%, preferably 5%).

[0181] This disclosure also provides a method for preparing polyclonal antibodies that specifically bind to any of the novel immunogens derived from general formula (1). Antisera containing the antibody can be obtained by a well-established technique in which animals such as rabbits and sheep are immunized with a suitable immunogen derived from general formula (1), and the antiserum is collected from the blood of the immunized animals after a suitable waiting period. Reviews include Parker, *Radioimmunoassay of Biologically Active Compounds* (Prentice-Hall, Englewood Cliffs, NJ, US, 1976), Butler, *J.Immunol.Meth.7:1~24 (1975)*, Broughton and Strong, *Clin.Chem.22:726~732 (1976)*, and Playfair Et al., *Br.Med.Bull.30:24~31 (1974)*. Immunoassay procedures are well-established in this field and are described in numerous professional books and publications, including "The Immunoassay Handbook, 4th Edition" (edited by David Wild, Nature Publishing Group, 2013) and the references cited therein. The degree to which antibody purification is necessary depends on the desired application. In many cases, purification is not necessarily required.

[0182] The collected serum can be tested for the presence of antibodies that specifically bind to MTX analyte using either an ELISA format or a homogeneous enzyme immunoassay format, using an MTX protein conjugate or other MTX conjugate. This technique is generally applicable to produce polyclonal antibodies against MTX analyte as described herein and to evaluate their usefulness. The prepared specific antibodies are useful as reagents for immunoassays for the detection or determination (including quantification, if necessary) of MTX.

[0183] The following procedure can be used to prepare monoclonal antibodies, particularly monoclonal antibodies that specifically bind to the immunogen of formula (1). Monoclonal antibodies can be manufactured according to the standard techniques of Kohler and Milstein (Nature 265:495-497, 1975). Reviews of monoclonal antibody techniques include Melchers et al., *Lymphocyte Hybridomas* (Springer-Verlag, New York, 1978), *Nature 266:495* (1977), *Science 208:692* (1980), and *Methods of Enzymology 73 (Part B):3-46* (1981). A sample of a suitable immunogen is injected into an animal such as a rabbit or mouse, and after a sufficient period of time, the animal is sacrificed to obtain spleen cells. Alternatively, spleen cells from an unimmunized animal can be sensitized to the immunogen in vitro. Splenocyte chromosomes encoding the target immunoglobulin sequence can usually be aggregated by fusing splenocytes with myeloma cell lines in the presence of a nonionic surfactant (e.g., polyethylene glycol). The resulting cells, including fused hybridomas, can be cultured in a selective medium such as HAT medium, and the surviving immortalized cells are subsequently cultured in that medium under limiting dilution conditions. The cells are cultured in a suitable container, such as a microtiter plate, and the resulting supernatant is screened for monoclonal antibodies with the desired specificity. Various techniques are known to increase the yield of monoclonal antibodies, for example, by injecting hybridoma cells into the peritoneal cavity of a mammalian host and collecting ascites fluid. If sufficient monoclonal antibodies do not accumulate in the ascites fluid, antibodies can be recovered from the host's blood. Alternatively, cells producing the target antibody can also be cultured using equipment known to those skilled in the art, such as hollow fiber cell culture devices or spinner flask devices. There are various known methods for separating and purifying monoclonal antibodies from other proteins and impurities (see Kohler and Milstein, cited above).

[0184] The following procedure can be used to prepare recombinant monoclonal antibodies, particularly monoclonal antibodies that specifically bind to the immunogen of formula (1). Single B cell screening, cloning, and expression were performed. Peripheral blood mononuclear cells (PBMCs) were isolated from whole rabbit blood, cultured on the same day, and seeded as single B cells in 40 × 96 well plates. These 40 × 96 well plates were cultured in B cell medium at 37°C / 5% CO2 for 7 days, and the supernatant was then screened by indirect ELISA for anti-BSA antigen to determine antigen-positive wells. Antigen-positive wells were stored in RNA lysis buffer and kept at -80°C. mRNA was isolated from the selected B cell wells (MTX-SH-BSA antigen-positive wells) using the Dynabeads mRNA DIRECT purification kit (Ambion, catalog number #61012). cDNA was synthesized, and two-step PCR was performed to prepare antibody variable region cDNA for cloning. Variable region cDNAs of rabbit IgG heavy chain and kappa light chain were cloned into mammalian expression vectors containing the constant regions of the rabbit heavy chain and light chain, respectively. The expression constructs were co-transfected into HEK293 cells, and the cell culture supernatant was assayed by indirect ELISA for DAMP-YS-BSA antigen. Antibodies were purified according to standard methods for antibody purification from the supernatant. Generally, antibodies can be purified by well-known techniques such as chromatography (e.g., DEAE chromatography, aBx chromatography, etc.) and filtration. Antibodies can be screened by several techniques, including using homogeneous enzyme immunoassay formats, taking into account characteristics such as conjugate inhibition, curve size, and cross-reactivity.

[0185] DNA sequencing was performed on the selected positive rabbit monoclonal antibodies. The rabbit IgG heavy chain sequence is approximately 1200 bp, and a reliable full-length variable region sequence can be obtained by sequencing from the 5' end. The rabbit kappa light chain is approximately 700 bp, and a reliable full-length variable region sequence can be obtained by sequencing from the 5' direction. All heavy chain and kappa chain variable region sequences were translated. The resulting example antibody heavy chain variable region (V H ) and light chain variable region (V L The amino acid sequence of ) is shown in Table 1 above, and the corresponding nucleotide sequence is shown in Table 2 above.

[0186] composition This disclosure also provides compositions. According to some embodiments, the compositions of this disclosure include any of the compounds, antibodies, nucleic acids, expression vectors and / or cells of this disclosure.

[0187] In certain embodiments, the compositions of the Disclosure comprise any of the compounds, antibodies, nucleic acids, expression vectors, and / or cells of the Disclosure present in a liquid medium. The liquid medium may be an aqueous liquid medium such as water or a buffer solution. Such compositions may contain one or more additives such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, surfactants (e.g., nonionic surfactants such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc.

[0188] In certain embodiments, the compositions of the Disclosure may be used as reagents for carrying out various immunoassays of the Disclosure, such as the homogeneous enzyme immunoassay described herein. For example, a composition comprising the compound of formula (1) of the Disclosure, the antibody of the Disclosure, or both may be used for carrying out such immunoassays. In some embodiments, the reagents may be provided in a lyophilized form, such as lyophilized reagent spheres, to enhance stability, convenience, etc. For example, a composition comprising the compound of formula (1) of the Disclosure, the antibody of the Disclosure, or both may be provided in the form of lyophilized reagent spheres, as described in U.S. Patent No. 5,413,732, the disclosure of which is incorporated herein by reference in whole. In short, a number of such spheres (reagent spheres) can be formed, for example, by preparing a homogeneous solution of the reagent, measuring a homogeneous drop (e.g., 2-50 μL) of the solution, dropping it dropwise into an unagitated cryogenic liquid (e.g., liquid nitrogen) and freezing it, recovering the frozen drop from the cryogenic liquid, and lyophilizing it.

[0189] Immunoassays Furthermore, aspects of the present disclosure include methods using the compound of formula (1) of the present disclosure and / or the antibody of the present disclosure. In certain embodiments, such compound and / or antibody may be used to detect (including determining the amount thereof) at least one MTX analyte in a medium, for example, a medium containing a biological sample of interest. For example, according to some embodiments, a method is provided for determining the amount of at least one MTX analyte in a medium, comprising combining a sample presumed to contain at least one MTX analyte with the antibody of the present disclosure in the medium. Such a method further includes determining the presence or absence of a complex containing the MTX analyte and the antibody, the presence of which indicates the presence of the MTX analyte in the sample. In certain embodiments, the medium may further comprise the compound of formula (1) of the present disclosure. For example, the medium may further comprise an MTX conjugate having an MTX moiety and a detectable label (e.g., an enzyme, G6PDH, etc.).

[0190] Any sample of interest may be a sample that is presumed to contain at least one MTX analyte. In certain embodiments, such samples include whole blood, serum, plasma, urine, sputum, semen, saliva, aqueous humor, cerebral fluid, cerebrospinal fluid, amniotic fluid, tissue culture media, and dilutions thereof.

[0191] This disclosure provides an immunoassay method for evaluating the presence or absence of MTX analyte in a sample presumed to contain analyte. The immunoassays of this disclosure may be in various formats. The immunoassay may be a segregation immunoassay (also called a heterogeneous immunoassay) or a homogeneous immunoassay. Furthermore, the immunoassay may be qualitative or quantitative. The assays of this disclosure include both sandwich assays and competitive assays. The immunoassay may embody other types of assays that are neither sandwich assays nor competitive assays, as in certain assays involving immunoprecipitation. In certain embodiments, the immunoassay is a homogeneous immunoassay, in which the assay reagent and the sample are mixed to form a homogeneous assay mixture.

[0192] In certain embodiments, the immunoassay is a homogeneous enzyme immunoassay system used for the analysis of MTX analytes in biological fluid samples. In some cases, the immunoassay is based on competition between MTX analytes in the sample and labeled MTX and / or MTX for an antibody binding site. In some embodiments, the label may be a protein such as an enzyme. For example, the label may be an enzyme whose activity can be measured by spectrophotometrics. As one non-limiting example, the assay of this disclosure uses MTX and / or acylated or alkylated DAMP labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for competition for an antibody binding site. In certain embodiments, enzyme activity decreases upon binding to the antibody, and as a result, the concentration of MTX analytes in the sample may be measured based on enzyme activity. In some cases, the active enzyme is nicotinamide adenine dinucleotide (NAD +It converts ) to NADH, resulting in an absorbance change that can be measured by spectrophotometrics. In certain cases, endogenous serum G6PDH does not inhibit immunoassays. This is because the coenzyme NAD + However, this is because it only functions with the enzyme of the bacterium (Leuconostoc mesenteroides) used in the assay.

[0193] Generally, the immunoassays of this disclosure may be carried out by adding (i) a sample presumed to contain MTX analite and (ii) an antibody specifically binding to MTX analite to a reaction mixture to detect the presence (or absence) of MTX analite in the sample, thereby forming a complex of the antibody, which may be present in the sample, with MTX analite. The method also includes detecting the presence or absence of the complex. The presence (or absence) of the complex may indicate the presence (or absence) of MTX analite in the sample. Furthermore, the amount of the formed complex may be evaluated to determine the concentration of MTX analite present in the sample (e.g., to provide an evaluation of the MTX analite concentration in the serum or tissue of the subject from which the sample was obtained). The presence and / or amount of the complex may be evaluated directly (e.g., by detecting the bound antibody in the complex) or indirectly (e.g., by evaluating the enzyme activity in the MTX enzyme conjugate). In this case, a detectable signal is generated when the MTX enzyme conjugate is not bound to the antibody, indicating that the MTX analyte antibody in the reaction mixture has bound to the MTX analyte derived from the sample (see, for example, Figure 14).

[0194] Generally, the immunoassays of this disclosure are performed in a medium (e.g., an assay medium or assay reaction mixture) in which a sample is combined with an MTX analyte antibody under conditions that allow for the formation of a stable complex between the analyte in the sample and the antibody.

[0195] The assay may be performed in solution or using any standard method (e.g., as described in Current Protocols in Immunology, Coligan Et Al., John Wiley & Sons, New York, 1992) with a solid (insoluble) carrier (e.g., polystyrene, nitrocellulose, particles, or beads). Such methods include ELISA (enzyme-linked immunosorbent assay), IRMA (immunoradioassay), and RIA (radioimmunoassay). In certain embodiments, the assay may be performed in solution, i.e., without the assay reagent being bound to or associated with a solid carrier.

[0196] If the assay is performed in solution, the test sample (and optionally a control sample) can be incubated with the anti-MTX analyte antibody for a sufficient time to form a complex with the analyte and affinity reagent. As previously mentioned, the anti-MTX analyte antibody may contain detectable labels such as radionuclides, fluorophors, or enzymes. The sample can then be processed to separate the MTX analyte antibody complex from the excess unreacted anti-MTX analyte antibody (e.g., by adding a secondary antibody (e.g., anti-immunoglobulin antiserum) followed by centrifugation to precipitate the secondary complex, or by binding to an affinity surface such as an immobilized unlabeled antibody, where the unlabeled antibody is immobilized on a solid substrate such as Sepharose® or a plastic well). Detection of the MTX analyte-bound MTX analyte antibody can be achieved by various methods. If necessary, a substrate corresponding to the detectable label may be added to the sample.

[0197] When the assay is performed using a solid support, the solid support may be one on which an MTX analyte antibody (or conjugate) is bound to the surface of the solid support. Binding of the assay reagent can facilitate the stable and wash-resistant binding of MTX analyte (or an antibody not bound to sample-derived MTX analyte, but present in the reaction mixture as in a competitive binding assay) to the solid support via specific binding to the antibody. The insoluble solid support may be any composition to which an antibody or suitable MTX conjugate can be bound, which is separable from soluble materials, and which is compatible with the overall method for detecting MTX analyte in the sample.

[0198] The surface of such a support may be solid or porous, and may have any shape. Examples of suitable insoluble solid supports to which MTX analyte antibodies or conjugates are bound include beads (e.g., magnetic beads), fluorescent particles, membranes, and microtiter plates. These may be composed of glass, plastic (e.g., polystyrene), polysaccharides, nylon, or nitrocellulose.

[0199] The assay reagent may include, in addition to the MTX analyte antibody disclosed herein, a detectable labeled secondary antibody as needed. After the assay reagent has bound to the solid support, the solid support may be treated with a blocking agent. Here, the blocking agent binds to the solid support in areas where the assay reagent has not bound. Suitable blocking agents include non-interfering proteins such as bovine serum albumin, casein, and gelatin. Alternatively, surfactants at non-interfering concentrations such as Tween, NP40, and TX100 may be used. Such blocking treatment can reduce nonspecific binding.

[0200] The assays described herein include both qualitative and quantitative assays. A typical quantitative method involves mixing analyte with a predetermined amount of reagent antibody and correlating the amount of the resulting complex with the amount of analyte in the original sample based on correlations derived using a standard sample containing a known amount of analyte within the expected range for the test sample. In a qualitative assay, the assay result can be determined if a sufficient amount of complex is obtained that exceeds or falls below a set threshold using a sample known to contain or not contain analyte. Unless otherwise specified, the acts of “measurement” or “determination” in this disclosure include both qualitative and quantitative determinations.

[0201] Immunosay reagents used alone or in combination in the assays described herein include, but are not limited to, MTX analyte-specific antibodies, MTX conjugates, and MTX analytes (e.g., as control samples or analytes in competitive binding assays). Immunosay reagents may be provided as buffered aqueous solutions. These solutions may further contain components such as surfactants, organic solvents, defoamers, buffers, surfactants, and preservatives. Surfactants may be added to retain hydrophobic or poorly soluble compounds in the solution and to stabilize components in the solution. Examples include bulk agents such as β-lactoglobulin (BLG) and polyethylene glycol (PEG), defoamers and surfactants such as Tween-20, Plurafac A38, Triton X-100, Pluronic 25R2, rabbit serum albumin (RSA), bovine serum albumin (BSA), and sugars. Examples of organic solvents include methanol and other alcohols. Various buffers may be used to maintain the pH of the solution during storage. Examples of buffering agents include HEPES, borates, phosphates, carbonates, Tris, and barbital. Preservatives also improve the storage stability of reagents for immunoassays.

[0202] The MTX analog DAMP conjugate and / or MTX analog analyte-specific antibody used as a reagent in the assays of this disclosure can be immobilized by binding to a solid support. This solid support may be, for example, the inner wall of a container containing the reagent, particulate matter, or a high molecular weight carrier that can be removed by physicochemical means such as centrifugation or microfiltration while maintaining a suspension state. In some cases, the binding occurs via one or more covalent bonds. The binding does not have to be covalent, but it should have sufficient strength and / or stability to withstand separation steps (including washing) that may be part of the assay procedure. In some cases, the solid support may be functionalized to include a reactive group to facilitate the binding of the MTX conjugate and / or MTX analog analyte-specific antibody to the solid support. Non-limiting examples of usable reactive groups include -COOH, -NH2, -C(O)H, and -SH. In some embodiments, the MTX analog conjugate and / or MTX analog analyte-specific antibody may be indirectly bound to the solid support by conjugating the protein carrier, which in turn may be conjugated to the solid support. Examples of particulate materials include, but are not limited to, agar, polystyrene, cellulose, polyacrylamide, latex particles, magnetic particles, and fixed red blood cells. Examples of commercially available matrices include, but are not limited to, Sepharose® (Pharmacia), Poros® resin (Roche Molecular Biochemicals), Actigel Superflow® resin (Sterogene Bioseparations Inc.), and Dynabeads® (Dynal Inc.). In certain embodiments, the selection of the solid support may be based on one or more of the following: stability, capacity, accessibility of the bound antibody, flow rate (or dispersibility of the resin in the reaction mixture), and ease of separation.

[0203] As described above, various formats may exist for immunoassays for the detection of MTX analytes. Generally, an immunoassay involves reacting one or more immunoassay reagents (e.g., at least an anti-MTX analyte specific antibody) with a test sample (i.e., a sample presumed to contain MTX analyte) in a medium (e.g., a reaction mixture or assay mixture). The “reaction mixture” or “assay mixture” generally refers to a mixed system formed by combining a sample presumed to contain MTX analyte with one or more immunoassay reagents, as illustrated herein, which enables the detection (qualitative or quantitative) of the presence or absence of MTX analyte in the sample. The reaction mixture is usually an aqueous solution, but the immunoassay reagents may be in solution or immobilized on a solid support (e.g., a substrate such as beads). The reaction mixture may contain other components suitable for the immunoassay, such as buffers and reagents.

[0204] Immunoassays are typically classified in several ways. For example, immunoassays can be classified based on their detection method, such as enzyme immunoassays, radioimmunoassays, fluorescence-polarized immunoassays, chemiluminescence immunoassays, and turbidity assays. Other classification methods are based on the assay procedure, such as competitive assay formats, sandwich assay formats, or assays based on the principle of precipitation or agglutination. In certain cases, they can also be further classified by whether or not a washing step is included (so-called heterogeneous assays), distinguishing them from those in which the reaction and detection are performed without a washing step (so-called homogeneous assays). Specific assays are described in more detail below.

[0205] Immunoassays can be classified as heterogeneous or homogeneous assays. As used herein, "homogenous immunoassay" refers to an assay method that detects the presence of a complex by utilizing the property that at least one of the reactants is incorporated into the complex, thereby gaining or losing the complex without separating the complex from the unreacted components. Homogeneous assays include systems using pairs of fluorochrome and fluorochrome quenchers arranged on different reagents, systems using pairs of enzymes and enzyme inhibitors, systems using pairs of chromophores and chromophore modifiers, and latex agglutination assays.

[0206] A specific homogeneous assay is a quantitative homogeneous enzyme immunoassay in which symmetric methylarginine is conjugated to an active enzyme. In certain embodiments, the conjugation may be designed so that an MTX analyte-specific antibody binds to the symmetric methylarginine conjugate, thereby qualitatively or quantitatively affecting the enzyme activity of the conjugate. When a sample containing MTX analyte is pre-mixed with the antibody, the antibody may form a complex with the MTX analyte, inhibiting its binding to the enzyme conjugate. In this way, the enzyme activity in the conjugate can be correlated with the amount of MTX analyte present in the sample.

[0207] G6PDH is a useful enzyme for this type of assay. In certain embodiments, G6PDH is a variant of the naturally occurring G6PDH, in which one or more lysine residues are deleted or substituted, or one or more cysteine ​​residues are introduced. For example, G6PDH from Leuconostoc mesenteroides is a dimeric enzyme, and NAD + or NADP + It has the ability to catalyze the oxidation reaction from D-glucose-6-phosphate to D-glucono-δ-lactone-6-phosphate using one of the following properties: NAD + The availability of NADP +Unlike human-derived G6PDH, which only utilizes human G6PDH, this method allows for the determination of L. mesenteroides-specific G6PDH activity even in the presence of human G6PDH. This is exemplified, for example, in human-derived samples. The two genera selected as sources of G6PDH are Leuconostoc and Zymomonas. Among these genera, L. mesenteroides, L. citreum, L. lactis, L. dextranicum, and Z. mobilis are of interest, with L. mesenteroides, L. citreum, and L. lactis being specific examples. Another example of a homogeneous assay system is the cloning enzyme donor immunoassay.

[0208] The MTX derivative DAMP-Y having a thiol-reactive group can be prepared as described above and may be reacted with a glucose-6-phosphate dehydrogenase (G6PDH) mutant to form the respective enzyme conjugates (see, for example, Figure 10). The mutant enzyme can be obtained by the methods described in U.S. Patents 6,090,567 and 6,033,890, which are incorporated herein by reference.

[0209] In some embodiments, the immunoassay may further include the step of adding a DAMP-Y MTX analog conjugate having an MTX moiety and a detectable label to the sample. The presence or absence of MTX analyte in the sample can be detected by detecting the detectable label. The detectable label may contain an enzyme, and the detection can be carried out by assaying the activity of the enzyme. In certain embodiments, the enzyme is a dehydrogenase, such as G6PDH.

[0210] The Luminescence Oxygen Channeling Immunoassay (LOCI) is a chemiluminescent homogeneous immunoassay that enables the detection of biotinylated analytes by having them compete for antibody binding sites on receptor beads. Biotinylated analytes bind to donor beads via streptavidin present on the donor beads. In the presence of analytes, the biotin-analyte binding interaction causes the two beads to be positioned in close proximity. When the donor beads are excited at 680 nm, singlet oxygen molecules are generated, which trigger a series of chemical reactions within the LOCI receptor beads, producing a detectable emission peak at 615 nm (Ullman, EF et al. (1996) Clin. Chem. 42, 1518-1526). When analytes are present, the amount of relative light units (RLU) emitted is inversely proportional to the concentration of the analyte.

[0211] In so-called "heterogeneous" assays involving a separation step, detection of the MTX analyte-specific antibody-analyte complex is performed by a process that includes physically separating the formed complex from the unreacted analyte, the unreacted antibody, or both.

[0212] In heterogeneous immunoassays, the antibody-MTX analyte complex is first formed in the liquid phase and then captured by a solid-phase reagent or separated based on changes in physical or chemical properties such as gel filtration or precipitation. Alternatively, one of the reagents may be immobilized on the solid phase before contact with the other, and the complex may be recovered after the reaction by washing away any unreacted reagent from the solid phase. Separation assays typically involve the use of labeled derivatives or labeled antibodies to facilitate the detection or quantification of the complex. Suitable labels include: 125Examples of labels include radioactive isotopes such as 1, enzymes such as peroxidase and β-galactosidase, and fluorescent labels such as fluorescein isothiocyanate. The separation step involves removing the labeling reagent present in the complex from the unreacted labeling reagent. The amount of labeling in the complex can be measured directly or indirectly estimated from the amount remaining unreacted.

[0213] The assays described herein include both sandwich assays and competitive assays. A sandwich assay generally involves the analyte to be measured forming a complex sandwiched between a reagent, such as a first antibody, which is ultimately used for the separation of the complex, and a reagent, such as a second antibody, which is used as a label for the separated complex. A competitive assay involves a system in which the analyte to be measured competes for binding with derivatives of the analyte and other reagents, such as antibodies. An example of a competitive assay using EMIT® is described in U.S. Patent No. 3,817,837.

[0214] The compounds and methods according to the embodiments of this disclosure also include the use of these materials in lateral flow chromatography techniques. Lateral flow chromatography is a technique that uses a membrane strip containing a detection device, such as a non-isotope signal-generating portion for MTX analytes. When a patient-derived sample is applied to the membrane strip, the sample interacts with the detection device and can produce results. These results can indicate multiple things, such as the absence or presence of MTX analytes in the sample, or their concentration.

[0215] In certain embodiments, a method is provided for qualitatively determining whether or not MTX analyte is present in a sample using lateral flow chromatography. In certain embodiments, the basic configuration of the qualitative lateral flow apparatus is as follows: (1) Sample pad: This is the part to which the sample is applied. The pad is treated with chemicals such as buffers and salts, which are redissolved to prepare the sample for reaction with conjugate reagents, test reagents, and control reagents. (2) Conjugate release pad: Generally composed of polyester or glass fiber material and treated with a conjugate reagent such as antibody-colloidal gold conjugate. A typical treatment method for the conjugate pad is impregnation followed by drying. When used, the liquid added as the sample redissolves the conjugate, which then flows into the membrane. (3) Membrane substrate: Usually composed of nitrocellulose or similar material, with the antibody-capturing component immobilized. (4) Wicking pad: Used in tests where plasma needs to be separated from whole blood. It is treated with reagents through impregnation to facilitate sample preparation and cell separation. (5) Absorbent pad: Functions as a reservoir for collecting liquid that has passed through the device. (6) Each of the above layers and membrane systems is laminated to an adhesive-backed plastic substrate that functions as a structural member.

[0216] In certain embodiments, a method is provided for qualitatively determining the presence of MTX analyte in a sample using lateral flow chromatography. In these embodiments, the membrane strip comprises a sample pad, which is a conjugate-releasing pad having an antibody specific to MTX analyte. This antibody may be conjugated to a non-isotope signal-generating moiety, such as colloidal gold particles. Other detection moieties useful in a lateral flow chromatography environment include dyes, colored latex particles, fluorescently labeled latex particles, and non-isotope signal-generating moieties. In some cases, the membrane strip further comprises a capture line, where the MTX analyte antigen or analyte conjugate is immobilized on the strip. In certain embodiments, this immobilization is optionally performed by covalent bonding to the membrane strip via a linking group. In other embodiments, immobilization may be performed by non-covalent bonding to the membrane strip. In yet another embodiment, the MTX analyte immobilized in the capture line is bound to a reactive partner, such as an immunogenic carrier like BSA.

[0217] A patient-derived sample is applied to a sample pad, where it binds to the antibody in the conjugate release pad, forming a solution. This solution chromatographically moves across the entire membrane by capillary action. If MTX analyte is present in the sample, an MTX analyte-antibody complex is formed and moves along the membrane by capillary action. When the solution reaches the capture line, the MTX analyte-antibody complex competes with insoluble MTX analyte for the limited antibody binding sites. If a sufficient concentration of MTX analyte is present in the sample, it will occupy the limited antibody binding sites. In certain cases, this inhibits the formation of colored antibody-insoluble MTX analyte complexes at the capture line. Therefore, the absence of color at the capture line indicates the presence of MTX analyte in the sample.

[0218] If MTX analyte is not present in the sample, a colored antibody-insoluble MTX analyte complex may form when the solution reaches the capture line of the membrane strip. In certain cases, the formation of this complex at the capture line is evidence that MTX analyte is not present in the sample.

[0219] In certain embodiments, a method is provided for quantitatively determining the amount of MTX analyte in a sample using lateral flow chromatography. This technique is further described in U.S. Patents 4,391,904, 4,435,504, 4,959,324, 5,264,180, 5,340,539 and 5,416,000, the disclosures of which are incorporated herein by reference. In certain embodiments, the antibody may be insolubilized over the entire length of the membrane strip. Generally, if the membrane strip is made of paper, the antibody may be covalently bound to the membrane strip. If the membrane strip is made of nitrocellulose, the antibody may be immobilized noncovalently to the membrane strip via hydrophobic or electrostatic interactions, etc. The membrane strip may include a conjugate release pad to which MTX analyte is bound to a detection portion. In certain embodiments, the detection portion is an enzyme, such as horseradish peroxidase (HRP).

[0220] In certain embodiments, a patient-derived sample is applied to a membrane strip, where it binds to the MTX analyte / detection molecule in the conjugate release pad, forming a solution. This solution chromatographically moves throughout the membrane by capillary action. If MTX analyte is present in the sample, both the MTX analyte and the MTX analyte / detection molecule compete for a limited number of antibody binding sites. If a sufficient concentration of MTX analyte is present in the sample, the limited number of antibody binding sites will be occupied. In certain cases, this will cause the MTX analyte / detection molecule to continue moving further within the membrane strip. A shorter distance traveled by the MTX analyte / detection molecule within the membrane strip indicates a lower concentration of MTX analyte in the sample, while a longer distance indicates a higher concentration. If the MTX analyte / detection molecule contains an enzyme, its travel distance can be detected by applying an enzyme substrate to the membrane strip. The detection of the enzyme reaction product allows for the determination of the MTX analyte concentration in the sample. In certain embodiments, an enzymatic chromogenic substrate, such as modified N,N-dimethylaniline, is insoluble in the membrane strip, and 3-methyl-2-benzothiazolinone hydrazone is passively applied to the membrane, thereby eliminating the need for a separate reagent to visualize the color reaction.

[0221] Fluorescence-polarized immunoassay (FPIA) technology is based on competitive coupling. FPIA technology is described, for example, in U.S. Patents 4,593,089, 4,492,762, 4,668,640, and 4,751,190, the disclosures of which are incorporated herein by reference.

[0222] FPIA technology can be used to identify the presence of MTX analytes and also in assays to quantify the amount of MTX analytes in a sample. This is partly based on the fact that the degree of polarization is proportional to the size of the molecule due to the rotational properties of the molecule in solution. Therefore, polarization can increase as the molecular size increases. That is, when a small molecule that rotates rapidly in solution, such as a fluorescently or otherwise luminescently labeled MTX analyte, is excited with linearly polarized light, the emitted light can be significantly depolarized. When a fluorescently labeled MTX analyte interacts with or binds to an antibody, its rotation slows down, and the emitted light can be highly polarized. In some cases, this is because the antibody significantly and measurably increases the size of the complex. Also, increasing the amount of unlabeled MTX analyte in a sample reduces the binding of the fluorescently labeled MTX analyte by the MTX analyte antibody, which reduces the polarization of the light emitted from the sample. The quantitative relationship between the concentration of unlabeled MTX analyte in a sample and its polarization can be established by measuring the polarization value by calibration using a known concentration of MTX analyte. Therefore, FPIA can be used to identify the presence and concentration of MTX analytes in a sample. WO95 / 16026 discloses a method for detecting MTX using FPIA and mouse IgG.

[0223] Homogeneous microparticle immunoassay technology (also known as immunoturbidimetric assay) is based on the aggregation of particles and compounds in a solution. When particles and / or chemical compounds aggregate, their particle size increases, and the turbidity of the solution increases. Therefore, by using MTX analyte antibodies in combination with microparticles, the presence of MTX analyte in a sample, and, if necessary, its amount, can be evaluated. Homogeneous microparticle immunoassays are useful because they can be performed on blood, hemolyzed blood, serum, plasma, tissue, and / or other samples. These assays can be configured to carry out the assay with MTX analyte supported on microparticles, or with MTX analyte antibodies supported on microparticles. Homogeneous microparticle immunoassays or immunoturbidimetric assays are used to measure the aggregation of substances in a sample. Immunoturbidimetric assay techniques are described, for example, in U.S. Patents No. 5,571,728, No. 4,847,209, No. 6,514,770, and No. 6,248,597, the disclosures of which are incorporated herein by reference. These types of assays utilize photoattenuation, nephelometry, or turbidimetric methods.

[0224] The Klond Enzyme Donor Immunoassay (CEDIA®, ThermoFisher) is based on competition between MTX analytes in a biological sample and MTX conjugates containing inactive recombinant enzyme donor (ED) fragments obtained from β-D-galactoside galactohydrolase or Escherichia coli-derived β-galactosidase ("β-gal"), which compete for binding to antibodies capable of binding to MTX analytes. ED containing thiols can be conjugated with the bromoacetamide of the present invention to generate an ED conjugate. If MTX analytes are present in the sample, the antibody binds to the MTX analytes, thereby restoring the enzymatic activity of the ED portion of the ED derivative conjugate to β-D-galactoside galactohydrolase or β-gal in the reaction mixture, making it capable of associating with enzyme acceptor (EA) fragments. This active enzyme, including ED and EA, can produce quantifiable reaction products when exposed to a suitable substrate. An example of a substrate is chlorophenol red-β-D-galactopyranoside (CPRG), which is hydrolyzed by the active enzyme to galactose and CPR, and CPR is measured by its absorbance at a wavelength of approximately 570 nm. If MTX analyte is not present in the sample, the antibody binds to the ED derivative conjugate, thereby inhibiting the association of the ED and EA fragments and preventing the recovery of enzyme activity. The amount of reaction product and the resulting change in absorbance are proportional to the amount of MTX analyte in the sample. Alternatively, a fluorescence signal can be obtained using a fluorescent substrate such as umbelliferone galactoside, or a luminescence signal can be obtained using a chemiluminescent substrate such as dioxetane galactoside or luciferin galactoside.

[0225] Competitive assays using chemiluminescent microparticle immunoassay (CMIA) technology can also be used to assess the presence or absence of MTX analyte in a sample (Regis Bouquie et al., Am J. Clin Path 2016). Various types of CMIA technology are available that can be used to determine the presence and / or amount of analyte in a sample. CMIA assays can utilize MTX analyte antibodies that can bind to MTX analyte, which can be bound to magnetic particles or particles separable by filtration, sedimentation, and / or other means. Furthermore, using tracers containing MTX or its derivatives bound to the appropriate chemiluminescent moiety, the binding of free MTX analyte in the patient sample to a limited amount of MTX analyte antibody on the particles can be made competitive. After the interaction of the sample, tracer, and antibody particles, the amount of tracer bound to the antibody particles is determined by chemiluminescence after a standard washing step to remove unbound tracer. Here, chemiluminescence is expressed in relative light units (RLU). The amount of chemiluminescence is inversely proportional to the amount of free analyte in the patient sample, and the concentration is determined by constructing a standard curve using known analyte values.

[0226] According to several embodiments, a homogeneous enzyme immunoassay is provided for analyzing MTX in biological fluids (e.g., whole blood, serum, plasma, urine, sputum, semen, saliva, lens fluid, cerebrospinal fluid, amniotic fluid, cultured tissue media, etc., and their dilutions). This assay is based on the competition between MTX present in biological fluids and an MTX analog DAMP labeled with an enzyme (e.g., glucose-6-phosphate dehydrogenase (G6PDH)) for antibody binding sites. Since enzyme activity decreases upon binding to an antibody, the MTX concentration in biological fluids can be measured as enzyme activity. For example, active G6PDH is nicotinamide adenine dinucleotide (NAD +The enzyme can be converted to NADH, and the resulting change in absorbance can be measured using a spectrophotometer. The assay can use an enzyme derived from bacteria (Leuconostoc mesenteroides), in which case the coenzyme NAD + Because it functions only with bacterial enzymes, endogenous serum G6PDH does not interfere.

[0227] In certain embodiments, one or more reagents are provided in lyophilized form for use in immunoassays of the Disclosure (including any homogeneous enzyme immunoassays described herein). As one example, a composition comprising the compound of formula (1) of the Disclosure, or the antibody of the Disclosure, or both, may be provided in the form of lyophilized reagent spheres (or "beads"), as described in U.S. Patent No. 5,413,732. The entirety of this Disclosure is incorporated herein by reference for all purposes. Briefly, the reagent spheres are formed, for example, by preparing a homogeneous solution of the reagents, measuring a homogeneous droplet (e.g., 2–50 μL) of the solution, dropping it dropwise into an unagitated cryogenic liquid (e.g., liquid nitrogen) to freeze, recovering the frozen droplet from the cryogenic liquid, and lyophilizing it.

[0228] According to some embodiments, for example, one or more reagents are provided in lyophilized form, and a centrifugal analyzer equipped with a microfluidic rotor (or “disk”) is used in the immunoassay of the present disclosure. For example, the immunoassay of the present disclosure may utilize an analyzer equipped with a centrifugal rotor for separating plasma from whole blood, the analyzer comprising multiple internal chambers and passages for mixing plasma or serum with one or more reagents (e.g., the aforementioned lyophilized spheres) and distributing the plasma or serum into multiple individual test wells. The chambers and passages required to separate whole blood into plasma are located radially outward from the metering chamber that supplies the volume of precisely metered blood and / or diluent to the separation chamber. The separation chamber includes a radially outward cell trap. As the rotor rotates, the cellular components of the whole blood are captured in the cell trap. The separated plasma is then delivered to multiple test wells or cuvettes. The above separation and dispensing steps typically occur as a result of the centrifugal force generated by the rotating rotor. The combination of the aforementioned lyophilized reagent spheres and the aforementioned rotor is particularly suitable for the analysis of plasma or diluted plasma. Furthermore, it is useful for a wide range of biological fluids, including urine, sputum, semen, saliva, lens fluid, cerebrospinal fluid, amniotic fluid, cultured tissue media, and even food and industrial chemicals. Details of this type of centrifugal analyzer equipped with a microfluidic rotor are described, for example, in U.S. Patents 5,061,381, 5,173,193, 5,122,284 and 5,186,844, the entirety of which is incorporated herein by reference for all purposes.

[0229] In some embodiments, the immunoassay utilizes a centrifugal analyzer that includes a microfluidic rotor equipped with a siphon for delivering a predetermined volume of liquid (e.g., a biological sample such as whole blood, serum, or plasma) between a first chamber and a second chamber within the rotor. The siphon may include a bend located radially inward from the radially innermost point of the liquid in the first chamber. While the rotor is rotating, the liquid does not flow beyond the bend. When the rotor stops, capillary forces draw the liquid around the bend, "priming" the siphon. When the rotor starts rotating again, centrifugal force draws the remaining liquid from the metering chamber to the receiving chamber, delivering it until the liquid level in the metering chamber reaches the same radial distance as the siphon outlet. The siphon may be designed such that the siphon inlet in the first chamber is radially outward from the siphon outlet in the second chamber. The positioning of the inlet and outlet offers certain advantages. For example, the siphon inlet is always positioned radially outward from the final position of the liquid meniscus after the liquid has been transferred from the first chamber to the second chamber. Therefore, measurement inaccuracies due to differences in meniscus shape in different liquids are suppressed by minimizing the meniscus. Furthermore, as those skilled in the art will understand, all siphons are metastable, and although the liquid column within the siphon is stable, it is easily broken when the rotor is disturbed. When the liquid column is broken, under centrifugal force, the liquid in the siphon flows to the radially outermost point. In conventional siphons, this point was the siphon outlet. Therefore, there was a possibility that an unmeasured volume of liquid would be delivered to the receiving chamber. In the siphon described herein, the radially outermost point within the siphon is the siphon inlet. This design avoids the problem of an unmeasured volume of liquid being delivered, as the liquid returns to the first chamber when the liquid column is broken. Further details regarding analytical apparatus including a centrifugal rotor with a siphon for delivering a predetermined volume of liquid, which may be used in the methods or immunoassays of this disclosure, are described in U.S. Patent No. 7,998,411, the entirety of which is incorporated herein by reference for all purposes.

[0230] The MTX derivatives, conjugates, antibodies, immunogens, and / or other conjugates described herein are applicable to other heterogeneous assays with various detection systems, including but not limited to enzymatic or fluorescent detection systems, as well as homogeneous immunoassays, including but not limited to rapid lateral flow assays and antibody arrays, and to forms that may be developed in the future.

[0231] While various immunodiagnostic assays utilizing the MTX derivatives, conjugates, antibodies, and immunogens described herein have been explained, these assays can also be modified. That is, within the scope of the embodiments described herein, various modifications can be made to the steps and operations for carrying out these immunoassays. Additional information related to assay formats can be found, for example, in David Wild's *The Immunoassay Handbook, 4th Edition* (Elsevier Science, published January 31, 2013).

[0232] KITS Embodiments of this disclosure also include kits. In some embodiments, such kits are used to determine the amount of at least one MTX analyte in a sample. Such kits may include the compound of formula (1) of this disclosure, or the antibody of this disclosure, or both.

[0233] In certain embodiments, a kit is provided for determining the amount of MTX analyte in a sample, such kit including the antibody of the Disclosure and instructions describing how to use the antibody to determine the amount of at least one MTX analyte. According to some embodiments, the antibody specifically binds to MTX with less than 0.1% cross-reactivity to 7-OH MTX. In certain embodiments, the kit containing the antibody of the Disclosure further includes a compound of formula (1) of the Disclosure. In some embodiments, the compound is a compound labeled Z, which is an enzyme such as glucose-6-phosphate dehydrogenase (G6PDH).

[0234] Furthermore, a kit is provided for determining the amount of at least one MTX analyte in a sample, the kit comprising any compound of formula (1) of the present disclosure and instructions for determining the amount of at least one MTX analyte using the compound. In certain embodiments, the compound is a compound labeled Z, such as an enzyme such as glucose-6-phosphate dehydrogenase (G6PDH). The kit may further comprise an antibody of the present disclosure. According to some embodiments, the antibody binds specifically to MTX, but has less than 0.1% cross-reactivity to the MTX metabolite 7-OH MTX, and the kit may further comprise instructions for determining the amount of MTX in a biological sample.

[0235] According to several embodiments, the kits of this disclosure are useful for conveniently performing assays to measure MTX analytes in a sample. Such kits may include (a) an antibody prepared to specifically bind to MTX and a compound of formula (1) as described herein (e.g., a DAMP conjugate), and instructions for measuring the amount of MTX analytes in a sample. In some embodiments, the kit further includes a conjugate of the compound of formula (1), wherein the conjugate includes a detectable label such as G6PDH. In certain cases, the kit may further include an auxiliary reagent for measuring the analytes. The antibody in the kit may be an antibody prepared against the compound of formula (1) as described herein.

[0236] To enhance the versatility of immunoassays, the reagents in a kit are provided in combination, packaged in liquid or lyophilized form in identical or separate containers, and the ratio of reagents may be configured to substantially optimize the method and assay. In certain embodiments, the kit comprises the antibody or compound of this disclosure, or both, and is provided as lyophilized reagent spheres as described in U.S. Patent No. 5,413,732 (the disclosure of which is incorporated herein by reference in its entirety for all purposes). The reagents contained in the kit may each be in a separate container, or multiple reagents may be combined in one or more containers depending on cross-reactivity and stability. In some embodiments, the compound of formula (1) described herein (e.g., MTX conjugate) exists in lyophilized form. In some embodiments, the antibody exists in lyophilized form. For example, the compound of formula (1) exists in a first lyophilized composition (which may further comprise one or more excipients, buffers, stabilizers, etc.), and the antibody exists in a second lyophilized composition (which may further comprise an enzyme substrate and one or more excipients, buffers, stabilizers, etc.). The first lyophilized composition and the second lyophilized composition may be provided as a single kit, for example, integrated in a single-use package or container.

[0237] The kit may further include other separately packaged reagents for performing the assay, such as co-enzyme substrates and other auxiliary reagents. The relative amounts of the various reagents in the kit can be varied widely to substantially optimize the reactions required when performing a method / immunoassay (e.g., a homogeneous enzyme immunoassay) and substantially optimize the sensitivity of the assay. Under appropriate conditions, one or more reagents in the kit may be provided as a dry powder, usually a lyophilized product, which may contain excipients and provide a reagent solution with a concentration suitable for performing the method or assay according to the present invention upon dissolution. The kit may further include a document describing the method according to the present invention as described above.

[0238] In the description of specific exemplary embodiments of the kit, the terms “and / or” are used to mean either the kit includes or does not include each item mentioned. This terminology is used for brevity. Generally, an immunoassay kit includes at least one antibody against an immunogen of an analyte, e.g., MTX, and at least one enzyme conjugate corresponding to an enzyme conjugate (e.g., a labeled conjugate) of the analyte, e.g., an MTX derivative.

[0239] In certain embodiments, an assay kit for MTX and / or MTX metabolites, which are analytes, is provided. The kit may include, as a package combination, (i) an antibody prepared against a compound of formula (1), and (ii) a conjugate of a derivative of the analyte. Another embodiment of the present disclosure is an assay kit for MTX and / or MTX metabolites, which includes, as a package combination, (i) an antibody prepared against a derivative of the analyte, and (ii) a conjugate of a hapten, which is a compound of formula (1).

[0240] The compounds, methods, and kits of this disclosure are used for routine monitoring by immunoassays. In certain embodiments, these immunoassays provide simple automated testing compatible with standard laboratory equipment and enable rapid turnaround time. To provide such immunoassays as described herein, antibodies against MTX are prepared. The derivatives and immunogens are designed, via the corresponding antibodies, to confer specific reactivity to MTX, cross-reactivity to 7-OH MTX of less than 0.1%, and detection capability of MTX at 0.03 micromol / liter.

[0241] The instructions included in this kit may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be included in the kit as a package insert or as labeling on the kit's container or its components (i.e., associated with the packaging or individual packaging). In other embodiments, the instructions may be provided as an electronic data file on a suitable computer-readable recording medium, such as a portable flash drive, DVD, CD-ROM, or diskette. In yet another embodiment, the actual instructions are not included in the kit, and a means of obtaining the instructions remotely, such as via the internet, is provided. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Similar to the instructions, the instruction retrieval means may be recorded on a suitable substrate or allow access to instructions for downloading or providing the instructions by scanning a QR code.

[0242] Notwithstanding the attached claims, this disclosure is also defined by the following clauses:

[0243] Article 1 The compound represented by the following formula (1): TIFF2026514355000016.tif33116 During the ceremony, R 1 is -YZ, Y is a linking group, Z is selected from the group consisting of hydrogen, OH, SH, S-acyl, O-alkyl, halogen, NH2, epoxy, maleidyl, haloacetamide, carboxyl, activated carboxyl, -N3 and alkene, immunogenic carrier, protein, label and solid support, and salts thereof.

[0244] Article 2 The compound according to Clause 1, wherein the linking group comprises 1 to 15 carbon atoms and / or 0 to 6 heteroatoms.

[0245] Article 3 The aforementioned linking group is -(CH2) n C(O)-, -C(O)(CH2) n -, -C(O)(CH2) n NHC(O)-, -C(O)(CH2) n NHC(O)(CH2) n -,-(CH2) n SCH2C(O)-, -(CH2) n C(O)NH(CH2) n -,-(CH2) n NHC(O)-,-(CH2) n NHC(O)(CH2) n -,-(CH2) n NHC(O)(CH2) n O(CH2) n NHC(O)(CH2) n -,-(CH2) n NHC(O)(CH2) n NHC(O)(CH2) n -, -NH(CH2) n C(O)-, -(CH2) n -, -C(O)NH(CH2CH2O) m (CH2) n NHC(O)(CH2) n -, and -(CH2) n (Heterocykrill) S(CH2) n A compound according to clause 1 or 2, selected from the group consisting of C(O)- and its salts, where each m is an independent integer between 1 and 10, and each n is an independent integer between 1 and 10.

[0246] Article 4 A compound as described in any one of clauses 1 to 3, wherein Z is a protein.

[0247] Article 5 The compound according to Clause 4, wherein the protein is an immunogenic carrier selected from the group consisting of hemocyanin, globulin, and albumin.

[0248] Article 6 The compound according to Clause 5, wherein the immunogenic carrier is bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).

[0249] Article 7 The compound according to Clause 1, wherein Z is an immunogenic carrier, and the immunogenic carrier is a polysaccharide.

[0250] Article 8 A compound described in any one of clauses 1 to 3, wherein Z is the label.

[0251] Article 9 The compound according to Clause 8, wherein the label is an enzyme.

[0252] Clause 10 The compound according to Clause 9, wherein the enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

[0253] Article 11 The compound according to clause 9, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).

[0254] Article 12 The compound according to any one of the clauses 1 to 11, wherein the linking group comprises an acyl group or alkyl group bonded to a phenyl group.

[0255] Article 13 The compound according to any one of the clauses 1 to 11, comprising an alkyl group bonded to a phenyl group or a substituted alkyl group.

[0256] Article 14 An antibody that specifically binds to a compound represented by formula (1) as described in any one of clauses 1 to 13.

[0257] Article 15 The aforementioned antibody V containing amino acid sequence (SEQ ID NO: 2) H CDR1, V containing amino acid sequence (SEQ ID NO: 3) H CDR2, and V containing amino acid sequence (SEQ ID NO: 4) H The heavy chain variable region (V) including CDR3 H ) polypeptides and, V containing amino acid sequence (SEQ ID NO: 6) L CDR1, V containing amino acid sequence (SEQ ID NO: 7) L CDR2, and V containing amino acid sequence (SEQ ID NO: 8) L Light chain variable region including CDR3 (V L ) polypeptides and, The antibody described in Clause 14, which competes for binding to the antibody and the compound.

[0258] Article 16 The antibody contains a heavy chain variable region (V) that has 70% or more identity with the amino acid sequence described in SEQ ID NO: 1. H The antibody described in Clause 15, comprising a polypeptide.

[0259] Article 17 The aforementioned antibody V containing amino acid sequence (SEQ ID NO: 10) H CDR1, V containing amino acid sequence (SEQ ID NO: 11) H CDR2, and V containing amino acid sequence (SEQ ID NO: 12) H The heavy chain variable region (V) including CDR3 H ) polypeptides and, V containing amino acid sequence (SEQ ID NO: 14) L CDR1, V containing amino acid sequence (SEQ ID NO: 15) L CDR2, and V containing amino acid sequence (SEQ ID NO: 16) L Light chain variable region including CDR3 (V L ) polypeptides and, The antibodies described in Clause 14, including those listed above.

[0260] Article 18 The aforementioned antibody A heavy chain variable region (V) containing an amino acid sequence that has 70% or more identity with the amino acid sequence described in SEQ ID NO: 9 H ) polypeptides and, Light chain variable region (V) containing an amino acid sequence having 70% or more identity with the amino acid sequence described in SEQ ID NO: 13 L ) polypeptides and, The antibodies described in Clause 17, including those listed therein.

[0261] Article 19 The aforementioned antibody V containing amino acid sequence (SEQ ID NO: 18) H CDR1, V containing amino acid sequence (SEQ ID NO: 19) H CDR2, and V containing amino acid sequence (SEQ ID NO: 20) H The heavy chain variable region (V) including CDR3 H ) polypeptides and, V containing amino acid sequence (SEQ ID NO: 22) L CDR1, V containing amino acid sequence (SEQ ID NO: 23) L CDR2, and V containing amino acid sequence (SEQ ID NO: 24) L Light chain variable region including CDR3 (V L ) polypeptides and, The antibody described in Clause 14, which competes for binding to the antibody and the compound.

[0262] Article 20 The aforementioned antibody A heavy chain variable region (V) containing an amino acid sequence that has 70% or more identity with the amino acid sequence described in SEQ ID NO: 17. H ) polypeptides and, Light chain variable region (V) containing an amino acid sequence having 70% or more identity with the amino acid sequence described in SEQ ID NO: 21 L ) polypeptides and, The antibodies described in Clause 19, including those listed therein.

[0263] Article 21 The antibody described in any one of the clauses 14 to 20, wherein the antibody is selected from the group consisting of IgG, Fv, single-chain antibody, scFv, Fab, F(ab')2, and Fab'.

[0264] Article 22 The antibody described in any one of the clauses 14 to 21, wherein the antibody is IgG.

[0265] Article 23 The antibody according to Clause 22, wherein the antibody is IgG1.

[0266] Article 24 The antibody described in any one of the clauses 14 to 21, wherein the antibody is a Fab.

[0267] Article 25 The antibody described in any one of the clauses 14 to 21, wherein the antibody is a single-chain antibody.

[0268] Article 26 The antibody described in Clause 25, wherein the antibody is scFv.

[0269] Article 27 The antibody described in any one of the clauses 14 to 26, wherein the antibody is a monoclonal antibody.

[0270] Article 28 The antibody described in Clause 14, wherein the antibody is a polyclonal antibody.

[0271] Article 29 The antibody according to Clause 28, wherein the antibody is a rabbit polyclonal antibody.

[0272] Article 30 The antibody according to any one of the claims 14 to 29, wherein the antibody further specifically exhibits 0.1% cross-reactivity with 7-hydroxymethotrexate.

[0273] Article 31 The heavy chain variable region (V) of the antibody described in any one of clauses 14 to 30. H) polypeptide, light chain variable region (V L ) A nucleic acid that codes for a polypeptide, or both.

[0274] Article 32 An expression vector comprising the nucleic acid described in Clause 31.

[0275] Article 33 Cells containing the nucleic acid of clause 31 or the expression vector of clause 32.

[0276] Article 34 The heavy chain variable region (V) of the antibody described in any one of clauses 14 to 30. H ) The first nucleic acid encoding the polypeptide, The light chain variable region (V) of the antibody described in any one of clauses 14 to 30. L ) The second nucleic acid that codes for the polypeptide, Cells that include this.

[0277] Article 35 A first expression vector containing the first nucleic acid, A second expression vector containing the second nucleic acid, Cells as described in Clause 34, including the cells described in Clause 34.

[0278] Article 36 A method for producing an antibody, comprising the step of culturing cells described in any one of the clauses 33 to 35 under conditions suitable for antibody expression, wherein the antibody is produced.

[0279] Article 37 A compound represented by formula (1) as described in any one of clauses 1 to 13, An antibody as described in any one of clauses 14 to 30, Nucleic acids as described in Article 31, The expression vector described in Clause 31, Cells as described in Clause 33 or 34, or A composition containing any combination of these.

[0280] Article 38 The composition according to Clause 37, wherein the composition is present in a liquid medium.

[0281] Article 39 The composition according to Clause 38, wherein the composition exists in a freeze-dried form.

[0282] Article 40 A method for determining the amount of methotrexate analyte in a medium, A step of contacting a sample presumed to contain at least one methotrexate analyte with an antibody described in any one of clauses 14 to 30 in a medium, A step of determining the presence or absence of a complex comprising the methotrexate analyte and the antibody, Includes, A method by which the presence of the complex indicates the presence of methotrexate analyte in the sample.

[0283] Article 41 The method according to Clause 40, wherein the medium further comprises a compound represented by formula (1) as described in any one of Clauses 1 to 13.

[0284] Article 42 The method described in Clause 41, wherein Z is a mark.

[0285] Article 43 The method according to clause 42, wherein the label is an enzyme.

[0286] Article 44 The method according to clause 43, wherein the enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

[0287] Article 45 The method according to clause 44, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).

[0288] Article 46 The method according to any one of the claims 40 to 45, wherein the determining step includes detecting the presence of an enzymatic reaction product of the compound.

[0289] Article 47 A kit for determining the amount of at least one methotrexate analyte in a sample, The antibody described in any one of clauses 14 to 30, Instructions for use for determining the amount of methotrexate analyzed in a sample using the aforementioned antibody, A kit that includes this.

[0290] Article 48 The kit according to Clause 47, wherein the antibody specifically exhibits less than 0.1% cross-reactivity with 7-hydroxymethotrexate.

[0291] Article 49 The kit described in Clause 47 further includes instructions for use for determining the amount of methotrexate with cross-reactivity of less than 0.1% to folic acid, folinic acid, dihydrofolate, and tetrahydrofolate.

[0292] Article 50 The kit according to Clause 47, wherein the antibody is present in a lyophilized form.

[0293] Article 51 A kit according to any one of clauses 47 to 50, further comprising a compound represented by formula (1) as described in any one of clauses 1 to 13.

[0294] Article 52 The kit described in Article 51, where Z is the marking.

[0295] Article 53 The kit according to Clause 52, wherein the label is an enzyme.

[0296] Article 54 The kit according to Clause 53, wherein the enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

[0297] Article 55 The kit according to Clause 54, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).

[0298] Article 56 The kit according to any one of clauses 52 to 55, wherein the compound is present in a lyophilized form.

[0299] Article 57 A kit for determining the amount of methotrexate analyte in a sample, A compound represented by formula (1) as described in any one of clauses 1 to 13, Instructions for using the compound to determine the amount of at least one methotrexate analyte in a sample, A kit that includes this.

[0300] Article 58 A kit as described in Article 57, with Z as the marking.

[0301] Article 59 The kit according to Clause 58, wherein the label is an enzyme.

[0302] Article 60 The kit according to Clause 59, wherein the enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

[0303] Article 61 The kit according to clause 60, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).

[0304] Article 62 The kit according to any one of the clauses 57 to 61, wherein the compound is present in a freeze-dried form.

[0305] Article 63 A kit according to any one of clauses 57 to 62, further comprising the antibody described in any one of clauses 14 to 30.

[0306] Article 64 The kit according to Clause 63, wherein the antibody specifically exhibits 0.1% cross-reactivity with 7-hydroxymethotrexate.

[0307] Article 65 The kit according to any one of clauses 57 to 64, wherein the antibody is present in a lyophilized form. [Examples]

[0308] The following examples are provided to those skilled in the art for a complete disclosure and explanation of the manufacturing method and use of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor to suggest that the experiments described below are all or only experiments. While efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors or deviations may be present. Unless otherwise specified, "parts" refers to parts by weight, molecular weight refers to weight-average molecular weight, temperature refers to degrees Celsius (°C), and pressure refers to atmospheric pressure or its vicinity. "Average" means the arithmetic mean. Standard abbreviations may be used, for example, bp: base pair, kb: kilobase, pl: picoliters, s or sec: seconds, min: minutes, h or hr: hours, aa: amino acid, nt: nucleotide, im: intramuscular, ip: intraperitoneal, sc: subcutaneous, etc.

[0309] The following abbreviations are used in relation to compounds, conjugates, and immunogens: DCM: dichloromethane, DMF: N,N-dimethylformamide, EDTA: ethylenediaminetetraacetic acid, KLH: keyhole-limpet hemocyanin, SATA: N-succinimidyl-S-acetylthioacetate, TFA: trifluoroacetic acid, EDCI: 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS: N-hydroxysuccinimide, DTT: dithioerythritol, G6PDH: glucose-6-phosphate dehydrogenase, Â: ethyl acetate, BSA: bovine serum albumin, MeCN: acetonitrile, t-Boc: tert-butyloxycarbonyl protecting group, TLC: thin-layer chromatography, MeOH: methanol, AcOH: acetic acid, PBST: phosphate-buffered saline containing Tween-20, TMB: 3,3',5,5'-tetramethylbenzidine, PBMC: peripheral blood mononuclear cells.

[0310] General Synthetic Procedures Many general references are available that provide well-known chemical synthesis schemes and conditions useful for synthesizing the disclosed compounds. (See, for example, Smith and March, *March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition* (Wiley-Interscience, 2001), Vogel, *A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, 4th Edition* (Longman, New York, 1978), and R.C. Larock, *Comprehensive Organic Transformations, 2nd Edition* (Wiley-VCH, 1999)).

[0311] The compounds described herein can be purified by any purification protocol well known to those skilled in the art, including chromatography such as HPLC, preparative thin-layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase can be used, such as conventional phase, reversed phase, or ion exchange resin. In certain embodiments, the disclosed compounds are purified by silica gel and / or alumina chromatography. See, for example, LRSnyder and JJ Kirkland, *Introduction to Modern Liquid Chromatography, 2nd Edition* (John Wiley and Sons, 1979), and E. Stahl, *Thin Layer Chromatography* (Springer-Verlag, New York, 1969). In terms of analytical techniques, LC-MS refers to liquid chromatography with mass spectrometry detection. 1 1H-NMR refers to proton nuclear magnetic resonance, which shows shifts in ppm units relative to tetramethylsilane (TMS).

[0312] In any step of preparing the compounds described herein, it may be necessary or desirable to protect sensitive or highly reactive groups in the molecules involved. Such protection can be achieved by using conventionally known protecting groups. Protecting groups are described in the following standard references: JFWMcOmie, *Protective Groups in Organic Chemistry* (Plenum Press, London and New York, 1973); TW. Greene and PGMWuts, *Protective Groups in Organic Synthesis, 3rd Edition* (Wiley, New York, 1999); E. Gross and J. Meienhofer (eds.), *The Peptides, Vol. 3* (Academic Press, London and New York, 1981); *Houben-Weyl Methoden der organischen Chemie, 4th Edition, Vol. 15 / 1* (Georg Thieme Verlag, Stuttgart, 1974); H.-D. Jakubke and H. Jescheit, *Aminosaurens, Peptides, Proteins* (Verlag Chemie, Weinheim, Deerfield Beach, and Basel, 1982); and Jochen Lehmann, *Chemie See "der Kohlenhydrate: Monosaccharide und Derivate" (Georg Thieme Verlag, Stuttgart, 1974). These protecting groups can be removed at an appropriate later stage using methods known to those skilled in the art.

[0313] The compounds described herein can be synthesized by various different synthetic schemes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. Various examples of synthetic schemes that can be used to synthesize the compounds disclosed herein are shown below.

[0314] Example 1: Synthesis of DAMP-1 hapten Step 1 TIFF2026514355000017.tif39158

[0315] Compound A [(2,4-diaminopteridine-6-yl)methanol hydrochloride, 5.0 g, 21.87 mmol] was dissolved in dry DMAC (85 mL), and Ph3PBr (27.7 g, 65.61 mmol) was added to this solution at room temperature and the mixture was allowed to react for 4 hours. Then, DIEA (12.7 g, 98.42 mmol) and compound B (4.5 g, 29.52 mmol) were added to this mixture at room temperature and the mixture was stirred overnight. The resulting reaction mixture was poured into a 0.33 M aqueous sodium hydroxide solution, and the precipitate was filtered. The pH of the filtrate was adjusted to 5.5 with 10% acetic acid, and the resulting precipitate was collected by filtration. After washing with water and diethyl ether, the mixture was dried overnight at 60°C to obtain orange solid DAMPA (4.9 g, 69.0%).

[0316] LCMS:R t =1.398 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 326.1, and the detected value is also 326.1.

[0317] Step 2 TIFF2026514355000018.tif49169

[0318] 4-[((2,4-diaminopteridine-6-yl)methyl)(methyl)amino]benzoic acid (DAMPA, 1.65 g, 5.072 mmol) was dissolved in dry DMSO (15 mL), to which TSTU (4.6 g, 15.22 mmol) and DIEA (2.0 g, 15.22 mmol) were added at room temperature and the mixture was allowed to react for 2 hours. Then, to this mixture, a solution of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl) carbamate (2.5 g, 10.14 mmol) dissolved in dry DMSO (10 mL) was added and the mixture was allowed to react overnight at room temperature. Water was added to this mixture and extracted with ethyl acetate (SiO). The organic layers were combined, dried over sodium sulfate (Na2SO4), and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2 / MeOH = 80 / 1~15 / 1, v / v) to obtain a crude orange solid. The crude product was recrystallized to obtain an orange solid (383 mg, 13.7%).

[0319] TLC: Rf=0.30 (silica gel, CH2Cl2 / MeOH=15 / 1, v / v) LCMS:R t =2.732 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 556.3, and the detected value is also 556.3.

[0320] HPLC:R t =9.016 minutes; area percentage (peak area ratio) 95% (214 nm); area percentage (peak area ratio) 98% (254 nm).

[0321] Step 3 TIFF2026514355000019.tif49170

[0322] A solution of tert-butyl(2-(2-(2-(4-[((2,4-diaminopteridine-6-yl)methyl)(methyl)amino]benzamide)ethoxy)ethoxy)ethyl)carbamate (383 mg, 0.6893 mmol) was mixed with HCl / dioxane (15 mL) and reacted overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and then H2O (30 mL) and NaOH (1.2 g, 30.0 mmol) were added and the mixture was treated at room temperature for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain an orange solid (252 mg, 80.3%).

[0323] LCMS:R t =3.205 minutes;[M+H] + The calculated value for m / z (mass-to-charge ratio) for this was 456.2, and the detected value was 456.3.

[0324] HPLC:R t =8.305 min; area percentage 98% (214nm); area percentage 98% (254nm).

[0325] Step 4 TIFF2026514355000020.tif59169

[0326] Example 2: Synthesis of DAMP-2 hapten Step 1 TIFF2026514355000021.tif28154

[0327] 3-(aminomethyl)aniline (40.0 g, 327.60 mmol) was dissolved in THF (660 mL), and Boc2O (38.7 g, 327.60 mmol) was added at 0°C and the mixture was reacted for 1 hour. The reaction mixture was concentrated to obtain a yellow solid (35.0 g, 48.0%).

[0328] LCMS:R t =2.553 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 223.1, and the detected value is also 223.1.

[0329] Step 2 TIFF2026514355000022.tif31151

[0330] 34.5 g, 155.2 mmol of tert-butyl(3-aminobenzyl)carbamate and 18.5 g, 155.2 mmol of benzotriazole were dissolved in ethanol (150 mL), and an aqueous formaldehyde solution (37%, 4.7 g, 155.2 mmol) was added at room temperature. The mixture was stirred overnight, and the solvent was removed by distillation. The residue was dissolved in THF (146 mL), and sodium borohydride (8.8 g, 282.8 mmol) was added at room temperature. The mixture was stirred for 4 hours, then saturated aqueous sodium bicarbonate solution and ethyl acetate were added, and extraction was performed. The organic layer was dried over sodium sulfate (Na2SO4) and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the title compound (17.0 g, 45% yield).

[0331] LCMS:R t =2.774 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 237.1, and the detected value is also 237.1.

[0332] Step 3 TIFF2026514355000023.tif35163

[0333] (2,4-diaminopteridine-6-yl)methanol hydrochloride (6.0 g, 26.03 mmol) was dissolved in dry DMAC (75 mL), and Ph3PBr (39.6 g, 78.12 mmol) was added at room temperature and allowed to react overnight. Then, DIEA (20.21 g, 98.42 mmol) and B (11.05 g, 39.06 mmol) were added to this mixture at room temperature and allowed to react overnight. The resulting mixture was poured into a 0.33 M aqueous sodium hydroxide solution, and the precipitate was filtered. The filtrate was adjusted to pH=5.5 with 10% acetic acid, and the resulting precipitate was collected by filtration. After washing with water and diethyl ether, it was dried overnight at 60°C to obtain an orange solid (4.8 g, 41.0%).

[0334] LCMS:Rt =3.083 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 411.3, and the detected value is also 411.3.

[0335] HPLC:R t =9.249 min; area percentage 79% (214 nm); area percentage 82% (254 nm).

[0336] Step 4 TIFF2026514355000024.tif28164

[0337] A solution of tert-butyl 6-(((3-(aminomethyl)phenyl)(methyl)amino)methyl)pteridine-2,4-diamine (2.1 g, 5.1 mmol) was reacted overnight with HCl / dioxane (50 mL) at room temperature. The resulting mixture was concentrated under reduced pressure, and then water (100 mL) and NaOH (4 g) were added at room temperature and the mixture was reacted for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (1.5 g, 94.3%).

[0338] LCMS:R t =0.909 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 312.2, and the detected value is also 312.2.

[0339] HPLC:R t =2.996 min; area percentage 100% (214nm); area percentage 100% (254nm).

[0340] Step 5 TIFF2026514355000025.tif63167

[0341] 8-((tert-butoxycarbonyl)amino)octanoic acid (828 mg, 1.6 mmol) was dissolved in dry DMSO (10 mL), to which TSTU (722 mg, 2.4 mmol) and DIEA (619 mg, 4.8 mmol) were added at room temperature and the mixture was allowed to react for 10 minutes. Then, 6-(((3-(aminomethyl)phenyl)(methyl)amino)methyl)pteridine-2,4-diamine (500 mg, 1.6 mmol) was added at room temperature and the mixture was allowed to react for 15 minutes. Water (100 mL) was added to the mixture at room temperature and the mixture was allowed to react for 5 minutes. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (500 mg, 56%).

[0342] LCMS:R t =3.103 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 552.4, and the detected value is also 552.4.

[0343] Step 6 TIFF2026514355000026.tif60151

[0344] A solution of tert-butyl[8-((3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzyl)amino)-8-oxooctyl]carbamate (500 mg, 0.9 mmol) was reacted overnight with HCl / dioxane (20 mL) at room temperature. The mixture was concentrated under reduced pressure, and then H2O (100 mL) and NaOH (4 g) were added at room temperature and reacted for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (280 mg, 68%).

[0345] LCMS:R t =3.492 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 452.3, and the detected value is also 452.3.

[0346] HPLC:R t =6.640 min; area percentage 100% (214nm); area percentage 100% (254nm).

[0347] Step 7 TIFF2026514355000027.tif75168

[0348] To a solution (0°C) prepared by dissolving the amino compound (745 mg, 1.61 mmol) in DMF (5 mL), 2,5-dioxopyrrolidine-1-yl-2-bromoacetate (454 mg, 1.93 mmol) was added. The resulting solution was stirred at room temperature for 3 hours. The reaction solution was then purified using Biotage Isolera One (eluted using a C18 column with 10%-90% MeCN / H2O (containing 0.1% TFA)), yielding the title compound DAMP-2 as a brown solid (360 mg, 62% yield).

[0349] MS:[M+H] + The calculated value for m / z (mass-to-charge ratio) for this was 572.5, and the detected value was 573.5.

[0350] Example 3: Synthesis of DAMP-3 hapten Step 1 TIFF2026514355000028.tif25154

[0351] To a solution of tert-butyl acrylate (10.1 g, 0.8 mol) and tert-butyl(4-hydroxybutyl) carbamate (10 g, 0.05 mol) dissolved in dioxane, 2 mL of 60% KOH aqueous solution was added, and the reaction mixture was stirred overnight at 25°C. The reaction was monitored by TLC, and KOH aqueous solution was added until the starting material, tert-butyl(4-hydroxybutyl) carbamate, was almost consumed. The reaction mixture was then mixed with DCM and washed three times with deionized water and once with saturated brine. The organic layer was collected, dried over Na₂SO₄, and the solvent was removed using a rotary evaporator. The resulting oily substance was purified by silica gel column with petroleum ether / ethyl acetate (10:1) to obtain a white oily Boc-protected amino alcohol (7 g, 43%).

[0352] Step 2 TIFF2026514355000029.tif17156

[0353] tert-butyl 3-(4-((tert-butoxycarbonyl)amino)butoxy)propanoate (5 g, 15 mmol) was dissolved in MeOH / H2O (15 mL / 15 mL) solution, and NaOH (1.3 g, 31 mmol) was added and the mixture was allowed to react overnight at room temperature. The reaction mixture was concentrated and used in the next step 3.

[0354] Step 3 TIFF2026514355000030.tif43168

[0355] 3-(4-((tert-butoxycarbonyl)amino)butoxy)propanoic acid (3.4 g, 13.2 mmol) was dissolved in dry DMSO (10 mL), to which TSTU (993 mg, 3.3 mmol) and DIEA (851 mg, 6.6 mmol) were added at room temperature and the mixture was allowed to react for 10 minutes. Then, 6-(((3-(aminomethyl)phenyl)(methyl)amino)methyl)pteridine-2,4-diamine (700 mg, 2.2 mmol) was added at room temperature and the mixture was allowed to react for 15 minutes. Water (100 mL) was added to the mixture at room temperature and the mixture was allowed to react for 5 minutes. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (700 mg, 58%).

[0356] LCMS:R t =2.901 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 554.4, and the detected value is also 554.4.

[0357] Step 4 TIFF2026514355000031.tif38167

[0358] A solution of tert-butyl [4-(3-((3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzyl)amino)-3-oxopropoxy)butyl]carbamate (500 mg, 0.9 mmol) was reacted overnight with HCl / dioxane (20 mL) at room temperature. The mixture was concentrated under reduced pressure, and then H2O (100 mL) and NaOH (4 g) were added at room temperature and reacted for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (310 mg, 77%).

[0359] LCMS:R t =0.892 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 454.3, and the detected value is also 454.3.

[0360] HPLC:R t =5.135 min; area percentage 100% (214nm); area percentage 100% (254nm).

[0361] Step 5 TIFF2026514355000032.tif41167

[0362] Deprotected 3-(4-aminobutoxy)-N-(3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzyl)propenamide (454 mg, 1.00 mmol) was dissolved in DMF (5 mL) (0°C), to which 2,5-dioxopyrrolidine-1-yl-2-bromoacetate (580 mg, 1.28 mmol) was added. The resulting solution was stirred at room temperature for 5 hours. The reaction solution was then purified using Biotage Isolera One (eluted using a C18 column with 10%-90% MeCN / H2O (containing 0.1% TFA)), yielding the title compound DAMP-3 as a yellow solid (260 mg, yield 45%).

[0363] LCMS purified product; MS:[M+H] + The calculated value for m / z (mass-to-charge ratio) for this was 574.5, and the detected value was 575.5.

[0364] Example 4: Synthesis of DAMP-4 hapten Step 1 TIFF2026514355000033.tif35168

[0365] 604 mg, 3.2 mmol of 3-((tert-butoxycarbonyl)amino)propanoic acid was dissolved in 10 mL of dry DMSO. 722 mg, 2.4 mmol of TSTU and 619 mg, 4.8 mmol of DIEA were added at room temperature and reacted for 10 minutes. Then, 500 mg, 1.6 mmol of 6-(((3-aminomethyl)phenyl)(methyl)amino)methyl)pteridine-2,4-diamine was added at room temperature and reacted for 15 minutes. 100 mL of water was added to this mixture at room temperature and reacted for 5 minutes. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (470 mg, yield 71%).

[0366] LCMS:R t =2.732 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 482.3, and the detected value is also 482.3.

[0367] Step 2 TIFF2026514355000034.tif34165

[0368] To a solution of tert-butyl(3-((3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzyl)amino)-3-oxopropyl)carbamate (470 mg, 0.9 mmol), HCl / dioxane (20 mL) was added at room temperature and the mixture was allowed to react overnight. The mixture was concentrated under reduced pressure, and then H2O (100 mL) and NaOH (4 g) were added at room temperature and the mixture was allowed to react for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (270 mg, yield 70%).

[0369] LCMS:R t =0.892 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 382.2, and the detected value is also 382.2.

[0370] HPLC:R t =3.968 min; area percentage 100% (214nm); area percentage 100% (254nm).

[0371] 1 H NMR:LQ-962-022, 400MHz, DMSO.

[0372] Step 3 TIFF2026514355000035.tif38168

[0373] 3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzyl)amino)-3-oxopropyl)amine (500 mg, 1.61 mmol) was dissolved in DMF (5 mL) (0°C), and 2,5-dioxopyrrolidine-1-yl-2-bromoacetate (454 mg, 1.93 mmol) was added to the solution. The resulting solution was stirred at room temperature for 3 hours. The reaction solution was then purified using Biotage Isolera One (eluted using a C18 column with 10%~90% MeCN / H2O (containing 0.1% TFA)), yielding the title compound DAMP-4 as a brown solid (258 mg, yield 24%).

[0374] LCMS:R t =0.963 minutes;[M+H] + The calculated m / z (mass-to-charge ratio) for this was 431.0, and the detected values ​​were 431.0 and 433.

[0375] HPLC:R t =10.253 min; area percentage 90% (214 nm); area percentage 91% (254 nm).

[0376] Example 5: Synthesis of DAMP-5 hapten Step 1 TIFF2026514355000036.tif43167

[0377] 6-(((3-aminomethyl)phenyl)(methyl)amino)methyl)pteridine-2,4-diamine (500 mg, 1.61 mmol) was dissolved in DMF (5 mL) (0°C), and 2,5-dioxopyrrolidine-1-yl-2-bromoacetate (454 mg, 1.93 mmol) was added to the solution. The resulting solution was stirred at room temperature for 3 hours. The reaction solution was then purified using Biotage Isolera One (eluted using a C18 column with 10%~90% MeCN / H2O (containing 0.1% TFA)), yielding the title compound as a brown solid (258 mg, yield 24%).

[0378] LCMS:R t =0.963 minutes;[M+H] + The calculated m / z (mass-to-charge ratio) for this was 431.3, while the detected values ​​were 431.0 and 433.

[0379] HPLC:R t =10.253 min; area percentage 90% (214 nm); area percentage 91% (254 nm).

[0380] Example 6: Synthesis of DAMP-6 hapten Step 1 TIFF2026514355000037.tif33168

[0381] 4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzoic acid (DAMPA) (1.65 g, 5.072 mmol) was dissolved in dry DMSO (15 mL). TSTU (4.6 g, 15.22 mmol) and DIEA (2.0 g, 15.22 mmol) were added to this solution at room temperature and the mixture was allowed to react for 2 hours. To the resulting mixture, tert-butyl-2-(2-aminoethyl)carbamate (1.7 g, 10.14 mmol) was dissolved in dry DMSO (10 mL) and the mixture was allowed to react overnight at room temperature. Water was added to the mixture and extracted with ethyl acetate (SiO). The organic layers were combined, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2 / MeOH = 80 / 1~15 / 1 (v / v)) to obtain a crude orange solid. The crude product was recrystallized to obtain an orange solid (383 mg, yield 13.7%).

[0382] TLC:R f =0.30 (silica gel, CH2Cl2 / MeOH=15 / 1, v / v).

[0383] Step 2 TIFF2026514355000038.tif30169

[0384] To a solution of tert-butyl(4-((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzoyl)amino)aminoethyl)carbamate (470 mg, 1.0 mmol), HCl / dioxane (20 mL) was added at room temperature and the mixture was allowed to react overnight. The mixture was concentrated under reduced pressure, water (100 mL) and NaOH (4 g) were added, and the mixture was reacted at room temperature for 2 hours. The resulting mixture was filtered, and the filtrate was freeze-dried to obtain a yellow solid (270 mg, yield 70%).

[0385] LCMS:R t =2.42 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 368.4, and the detected value is also 368.4.

[0386] HPLC:R t=3.968 min; area percentage 100% (214nm); area percentage 100% (254nm).

[0387] Step 3 TIFF2026514355000039.tif44168

[0388] 3-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzoyl)amino)ethylamine (500 mg, 1.36 mmol) was dissolved in DMF (5 mL) (0°C), and 2,5-dioxopyrrolidine-1-yl-2-bromoacetate (384 mg, 1.63 mmol) was added to the solution. The resulting solution was stirred at room temperature for 3 hours. The reaction solution was then purified using Biotage Isolera One (eluted using a C18 column with 10%~90% MeCN / H2O (containing 0.1% TFA)), and the title compound was obtained as a brown solid (318 mg, yield 48%). LCMS:R t =5.83 minutes;[M+H] + The calculated value of m / z (mass-to-charge ratio) for this is 489.3, and the detected value is also 489.3.

[0389] Example 7: Preparation of DAMP-YS-BSA conjugate Lysine residues in BSA were thiolated by reacting them with N-succinimidyl-S-acetylthioacetate (SATA) in carbonate buffer (pH 9.0). Hydrolysis with hydroxylamine yielded thiolated BSA (BSA-SH), which was then reacted with DAMPA-Y-bromoacetamide to obtain the DAMP-YS-BSA conjugate. The resulting conjugate was purified by dialyzing with 10kD Slide-A-Lyzer™ MWCO in 2 L of phosphate buffer (pH 7.4) twice.

[0390] The DAMP-YS-BSA conjugate was used for screening B cells and monoclonal antibodies to screen antibodies obtained from rabbit blood samples using indirect ELISA (Figure 12).

[0391] Example 8: Preparation of DAMP-YS-KLH immunogen The hapten DAMP-Y (compounds 1-6 shown above) was conjugated to KLH to which a thiol group had been chemically introduced. N-succinimidyl-S-acetylthioacetate was reacted with the primary amine of KLH to introduce a protected sulfhydryl group. Subsequently, the protected sulfhydryl group was deprotected with hydroxylamine to obtain the desired thiolated SH-KLH (Figure 11). Conjugation of DAMP bromoacetamide hapten with SH-KLH yielded the immunogen DAMP-YS-KLH (Figure 11).

[0392] A) Preparation of SH-KLH Lyophilized KLH (20 mg) was redissolved in deionized water, and the pH was adjusted to 8.6 with 1.0 M sodium bicarbonate-sodium carbonate buffer. A solution of N-succinimidyl-S-acetylthioacetate (4.67 mg dissolved in 92 μL of DMF to prepare a 220 mM solution) was prepared and gradually added to the KLH solution over 4 hours. The reaction mixture was stirred at room temperature during the addition of N-succinimidyl-S-acetylthioacetate, and then stirred for a further 16 hours in a cold room at 4°C.

[0393] Deacetylation to generate sulfhydryl groups for cross-linking was performed by adding 200 μL of a deacetylation solution (0.7 M hydroxylamine dissolved in 12.5 mM NaH2PO4-Na2HPO4 buffer (pH 7)). The contents were mixed, and the reaction mixture was incubated at room temperature for 2 hours to obtain SH-KLH (see Figure 11). At the end of this reaction, EDTA was added to a final concentration of 1 mM.

[0394] B) Preparation of DAMP-YS-KLH immunogen To minimize disulfide bond formation, dithiothreitol solution was added to the SH-KLH solution to achieve a final concentration of 1 mM. The pH was adjusted to 7.2 with 1 M sodium bicarbonate-sodium carbonate buffer. 146 μL of hapten DAMP-L-bromoacetamide (9.6 mg / 0.2 mL) dissolved in DMF was gradually added to the SH-KLH solution over 4-5 hours. The reaction was allowed to continue overnight at 4°C. The resulting mixture was dialyzed using a 10,000 MWCO Slide-A-Lyzer® dialysing cassette (Pierce) with 2 liters of 12.5 mM NaH2PO4-Na2HPO4 buffer (pH 7.0) twice at 2-8°C. This procedure yielded immunogen DAMP-YS-KLH.

[0395] Example 9: Preparation of DAMP-SH-G6PDH conjugate The DAMP-bromoacetamide haptens prepared as described in Examples 1-6 are designed to be compatible with proteins containing cysteine ​​groups (e.g., mutant G6PDH (U.S. Patents 6,455,288, 6,090,567, and 6,033,890)) or G6PDH to which thiol groups have been introduced by chemical reaction as described in Example 8.

[0396] DAMP hapten (0.02 mmol) was dissolved in DMF (0.21 mL). This solution was stirred at room temperature for 30 minutes. This DAMP solution was used as follows.

[0397] To reduce the cysteinethiol group forming the disulfide bond to a sulfhydryl group, DTT solution was added to mutant G6PDH to a final concentration of 2 mM. The pH of the resulting enzyme solution (0.9 mg, 1.5 mL) was adjusted to 7.2 with 1 M sodium bicarbonate-sodium carbonate buffer and mixed with DAMP-bromoacetamide hapten (0.07 mL, approximately 340-fold molar excess). The reaction mixture was gently stirred at 4°C for 16 hours. The excess DAMP-bromoacetamide hapten was separated from the enzyme-hapten conjugate by passing the reaction mixture through a Sephadex G-50 column in 12.5 mM NaH2PO4-Na2HPO4 buffer (pH 7.0). The column elution fraction containing the enzyme-hapten conjugate was identified by measuring the absorbance at 280 nm, and these were collected to obtain conjugated DAMP-YS-G6PDH (see Figure 10).

[0398] Example 10 Preparation of polyclonal antibodies against MTX Twenty-four New Zealand white female rabbits were immunized by subcutaneous administration of 200 μg per rabbit of the DAMP-YS-KLH immunogen prepared in Example 8, emulsified in complete Freund's adjuvant. After the initial administration, the same immunogen was emulsified in incomplete Freund's adjuvant and administered at a dose of 100 μg per rabbit every four weeks. 134 days after the initial immunization, blood samples containing polyclonal antibodies were obtained from the central artery of the ear of each rabbit. Antiserum (containing DAMP-YS-KLH antibodies) obtained from these blood samples were evaluated in a homogeneous assay format by measuring maximum antibody inhibition against the enzyme conjugate DAMP-YS-G6PDH and modulation in the presence of MTX. Based on these experimental results, rabbits 21342 and 26494 were selected from among rabbits immunized with the DAMP-YS-KLH immunogen to isolate peripheral blood mononuclear cells (PBMCs) as a source of B cells for cloning. Rabbit polyclonal antisera from 24 rabbits (samples) immunized with DAMP-Y-SH-KLH were screened, and clones were selected that showed the maximum separation (ΔmA / min) in the MTX assay and the least cross-reactivity to 7-OH-MTX, trimethoprim, triatelene, and the folate analogs shown in Figure 3.

[0399] Example 11 Preparation of rabbit monoclonal antibody To efficiently sample the natural antibody repertoire of immunized rabbits, recombinant rabbit monoclonal antibodies were prepared using a single B-cell screening strategy. This technique is generally applicable to the production of monoclonal antibodies against MTX, as described herein.

[0400] Rabbit peripheral blood mononuclear cells (PBMCs) obtained from immunized animals #21342 and #26494 were used as a source of B cells. Approximately 40 mL of whole blood was collected from each rabbit, and PBMCs were isolated by standard density gradient centrifugation. The obtained PBMCs were suspended in PBS and dispensed into 40 96-well plates on the same day for culture, theoretically resulting in one cell per well.

[0401] The supernatant obtained from each well was tested by indirect ELISA for DAMP-YS-BSA antigen. Forty 96-well microtiter plates were coated with DAMP-YS-BSA (0.1 μg / well) in 0.1 M carbonate buffer (pH 9.5) and left to stand overnight at 4°C. The plates were emptied, shielded with 3% skim milk powder in PBST, and shaken at room temperature for 1 hour. After removing the shielding solution, the plates were rinsed with PBST. 25 μL of PBST was added to each well, followed by 25 μL of cell supernatant in all wells, and the cells were incubated in a 37°C incubator for 1 hour. The plates were washed five times with PBST for a total washing time of 30 minutes. Subsequently, a 1:10000 (v / v) dilution of the secondary antibody, goat anti-rabbit IgG-Fc-HRP conjugate, was added at a rate of 100 μL / well in PBST, and the plates were incubated at 37°C for 1 hour with constant shaking. The plate was washed five times with PBST (total 30 minutes). Subsequently, 50 μL / well of TMB substrate was added. After allowing the color to develop in the dark for 5 minutes, the reaction was stopped by adding 50 μL of 1N HCl. The color development was measured using a microplate reader at 450 nm, and the obtained data was transferred to a computer for analysis. The supernatant that showed color development in the wells, i.e., bound to the DAMP-Y-SH-BSA conjugate, was determined to be positive. From these, 32 cells were selected for cloning and expression.

[0402] For each ELISA well containing antigen-specific antibodies, mRNA was isolated from the corresponding PBMC culture well, and this was divided to separately synthesize cDNA derived from the variable regions of the rabbit genes IgH and IgK, respectively. After two-step PCR amplification, the resulting cDNA was seamlessly ligated into separate mammalian expression vectors having either a constant IgG region in the heavy chain or a constant IgK region in the light chain. The ligation mixture was transformed into E. coli, the correct expression construct was selected and cultured, and the plasmid was isolated. The expression construct was co-transfected into HEK293 cells. The transfected cells were cultured for 2 days to secrete recombinant antibodies. The antigen-binding ability of the recombinant expression antibodies was evaluated by indirect ELISA against the DAMP-YS-BSA antigen as described above. The selected clones were further used for evaluation in the homogeneous enzyme immunoassay format described in Examples 12 and 15. DNA sequencing was performed on the selected rabbit monoclonal antibodies, and the data was translated based on standard codes to obtain protein sequence data for all heavy chain and κ chain variable regions. The table below summarizes the steps involved in obtaining the monoclonal antibodies.

[0403] [Table 3]

[0404] Example 12: Reagent and Assay The MTX antibody and enzyme conjugates of this disclosure can be advantageously used to detect MTX analytes in a sample in a homogeneous assay format. Antibodies can be evaluated by known methods such as conjugate inhibition, conjugate modulation, calibration, cross-reactivity, and spike recovery. For this purpose, antibody reagents are prepared by adding cloned antibodies (28H3-K1, 32H1-K2, 64H1-K1) to antibody diluent. In addition to the antibodies prepared above, the antibody reagents include buffer, salts, stabilizers, preservatives, and NAD. + It also contains glucose-6-phosphate. The enzyme conjugate reagent is prepared by adding the enzyme conjugate DAMP-YS-G6PDH to the conjugate diluent. The enzyme conjugate reagent contains the conjugate, buffer, stabilizer, salts, and preservative.

[0405] The Beckman Coulter AU480 (Blair, CA, USA) is a useful clinical chemistry analyzer for evaluating antibody and enzyme conjugates in a homogeneous enzyme immunoassay format. The Beckman AU480 is an automated biochemistry spectrophotometer analyzer used in medical laboratories to process biological fluid samples such as urine, cerebrospinal fluid, oral fluid, plasma, and serum. This analyzer can maintain a constant temperature, pipette samples, mix reagents, measure absorbance, and accurately measure reaction times.

[0406] Homogeneous enzyme immunoassays are performed using a ready-to-use two-reagent liquid assay prepared as described above. Typically, 2-15 μL of sample containing MTX analyte is incubated with 75-150 μL of antibody reagent, and then 50-100 μL of enzyme conjugate reagent is added.

[0407] This assay is a homogeneous enzyme immunoassay technique used to analyze MTX in biological fluids. This assay is based on competition for the antibody binding site between MTX in the sample and MTX or an MTX analog (the DAMP derivative of this invention) labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Since enzyme activity decreases upon antibody binding, the drug concentration in the sample can be measured based on enzyme activity. The active enzyme is nicotinamide adenine dinucleotide (NAD). + This converts to NADH, resulting in a change in absorbance at 340 nm, which can be measured by a spectrophotometer. Endogenous serum G6PDH does not interfere, but this is due to the coenzyme NAD + However, this is because it functions only with the bacterial (Leuconostoc mesenteroides) enzyme used in this assay. The change in absorbance at 340 nm can be measured by spectrophotometrics, which is proportional to the enzyme conjugate activity, and this activity is further related to the analyte concentration (see Figure 14).

[0408] Example 13: Antibody and calibration using MTX analyte MTX analyte antibodies and enzyme conjugates (DAMP-YS-G6PDH) were used to create calibration curves using MTX standards in a homogeneous assay format, as described in Example 12. Antibody reagents were prepared using immunorabbit antibodies generated by DAMP-Y-KLH and antibodies cloned via B cells, as described in Examples 8-11. The enzyme conjugate DAMP-YS-G6PDH was used to prepare the conjugate reagents. Calibration curves were created on a Beckman AU480 clinical chemistry analyzer, as described in Example 12. These experiments confirmed that the polyclonal antibodies rabbit #21342, rabbit #26494, rabbit #27420, and cloned monoclonal antibodies exhibited antibody-binding reactions to MTX and showed dose-response relationships.

[0409] Approximately 30 mL of heparinized whole blood was collected from rabbits #25149 (MT-1) and #25779 (MT-2), and peripheral blood mononuclear cells (PBMCs) were isolated and cultured from this whole blood. In vitro culture of B cells (40 × 96 wells per rabbit) was performed, and the supernatant was screened for MTX-BSA antigen by indirect ELISA. 240 antigen-positive B cell candidates were obtained from rabbit #25149, and 27 antigen-positive B cell candidates were obtained from rabbit #25779. 30 antigen-positive B cells were selected from rabbit #25149, and 6 antigen-positive B cells were selected from rabbit #25779 for cloning, expression, and further screening. In short, mRNA was isolated from the 36 selected B cells, cDNA was synthesized, and two-step PCR was performed to prepare antibody variable region cDNA for cloning. The cDNAs of the rabbit IgG heavy chain variable region and the κ light chain variable region were cloned into mammalian expression vectors containing the rabbit heavy chain constant region and the rabbit light chain constant region, respectively. The expression constructs were co-transfected into HEK293 cells, and the cell culture supernatant was assayed by indirect ELISA for MTX-BSA antigen. Thirty-six B cells were successfully cloned and expressed in HEK293 culture medium. Of the 36 successfully expressed B cells, ten clones were selected and advanced to the 20 mL stage (Table 4) to produce large quantities of recombinant rabbit antibody for further characterization.

[0410] [Table 4]

[0411] The top-performing clones (28H3 / 28K1, 32H1 / 32K2, and 64H1 / 64K1) were selected based on their performance in large-scale production and purification (Table 5). These clones were then used for further research. To prepare the initial antibody reagent, both clones were diluted to 100 μg / mL with antibody diluent. Then, clones 32H1 / 32K2 and 64H1 / 64K1 were mixed in a 1:4 ratio and added to the antibody diluent. To prepare the initial antibody reagent, both clones were diluted to 100 μg / mL with antibody diluent. Then, clones 32H1 / 32K2 and 64H1 / 64K1 were mixed in a 1:4 ratio and added to the antibody diluent.

[0412] [Table 5]

[0413] Example 14: Antibody and calibration using MTX A calibration curve was created using a standard prepared with MTX according to the method described in Example 7, using an MTX analyte antibody and an enzyme conjugate (DAMP-YS-G6PDH) in a homogeneous assay format. Antibodies derived from clone 64H1-K1 were used to prepare the reagent and to detect MTX in a homogeneous assay format. MTX was quantified based on the calibration curve obtained from the standard prepared with MTX (see Figure 14). The enzyme conjugate DAMP-S-G6PDH was used to prepare the conjugate reagent. The calibration curve was prepared on a Beckman AU480 clinical chemistry analyzer as described in Example 8. Typical calibration curves are shown in the table below, and dose-response curves are shown in Figure 14. The spike recovery test was performed as described above, and the results are shown in Table 9.

[0414] Calibration curve The calibration range for the MTX assay is 0.00 to 1.30 μM. A calibration curve was created on the Beckman AU680 by performing quadruple measurements at each calibrator level. The Beckman AU680 analyzer automatically performs nonlinear data analysis using a multi-parameter curve-fit math model. Typical calibrator separations in the MTX assay are shown in Table 6. Figure 2 shows the calibration curve graph for the feasibility lot reagent, and the percent modulation was calculated to be approximately 88.8% based on a maximum rate of 640.3 mA / min. The formula for calculating the percent modulation in the feasibility lot is as follows.

[0415] ·Max Rate=640.3mA / min • Negative calibrator rate = 358.5 mA / min • High calibrator speed (1.30 μM) = 608.7 mA / min • The entire curve ΔmA = high calibrator velocity - negative calibrator velocity (608.7 - 358.5 = 250.2 mA / min) Modulation rate (%) = Total curve ΔmA ÷ (Maximum velocity - Negative calibrator velocity) × 100 ·250.2mA / min÷(640.3-358.5mA / min)×100=88.8%

[0416] [Table 6]

[0417] [Table 7a]

[0418] [Table 7b]

[0419] [Table 7c]

[0420] Example 15: Spike-Recovery Known amounts of MTX analyte stock solution (mM) were added (spiked) to the synthetic matrix to prepare concentrations of 0, 0.06, 0.10, 0.30, 0.60, and 1.20 mmol / L. These samples were triple-repeated using homogeneous enzyme immunoassay on a Beckman AU480 as described in Example 8 to confirm the concentration (recovery rate) of the spiked samples. The samples were quantified using a series of separately prepared standards to create a six-calibration curve. The calibration curve was created using standards prepared with the same analyte as the analyte quantified in the test sample. The enzyme conjugate reagent used was a conjugate containing DAMP-YS-G6PDH, and the antibody reagent was derived from clones 28H3-K1, 32H1-K2, or 64H1-K1. The enzyme conjugate reagent contained a DAMP-YS-G6PDH conjugate, and the antibody reagent was derived from clones 28H3-K1, 32H1-K2, or 64H1-K1. The MTX analyte concentration recovered in the spike recovery test was compared to known concentrations.

[0421] In another experiment, the same sample as described above was subjected to triple replication by homogeneous enzyme immunoassay and double replication by LC-MS-MS for quantification. Deuterated MTX was used as an internal standard in the LC-MS-MS method. The homogeneous enzyme immunoassay was prepared using clone 32H1-K2 or 64H1-K1 in the antibody reagent and the DAMP-YS-G6PDH conjugate in the enzyme conjugate reagent. The results showed that this homogeneous enzyme immunoassay quantified MTX levels in a manner consistent with LC-MS-MS (see Figure 15).

[0422] In another experiment, a known amount of MTX was analyzed by homogeneous enzyme immunoassay, and the concentration (recovery rate) of the spike sample was confirmed on a Beckman AU480 as described in Example 12. Calibration curves were prepared using standards prepared with the same analyte as the analyte quantified in the test sample. The enzyme conjugate reagent contained the DAMP-YS-G6PDH conjugate, and the antibody reagent was prepared to contain antibody clones 32H1-K2 and / or 64H1-K1. The MTX analyte concentrations recovered in the spike-recovery test were compared with the known concentrations, and the results are shown in Table 9.

[0423] Example 16 Tables 8a, 8b, and 8c - Precision Study results Calibration curves were created, and 10-fold duplicate measurements were performed for each quality control (QC) sample. This procedure was repeated to obtain 20-fold duplicate measurements for each control level. The root mean square (RMS SD) was calculated for each control level. As shown in Table 8, the %CV of the MTX assay ranged from 1.48% to 2.85% for all QC levels tested.

[0424] [Table 8a]

[0425] Accuracy evaluation (overall accuracy or laboratory accuracy) was performed in accordance with CLSI guidelines. This study involved two runs per day over 20 non-consecutive days, with quadruple measurements per run for each level. A calibration curve was created during these 20 days. All quality control (QC) levels and corresponding pooled human serum control samples were tested for each measurement. The pooled serum sample was used to demonstrate equivalent accuracy in both the synthetic calibrator / control matrix and human serum.

[0426] Tables 8b and 8c summarize the accuracy of all control samples and patient serum pools over a 20-day period, with overall accuracy coefficients ranging from 1.40% to 3.00%.

[0427] [Table 8b]

[0428] [Table 8c]

[0429] Analysis recovery rate Test samples were prepared by spike-injecting negative pooled human serum with MTX at concentrations of 0.060, 0.100, 0.300, 0.600, 1.000, and 1.200 μmol / L. Table 9 shows the results for each level. Recovery rates ranged from 104.4% to 107.9%.

[0430] [Table 9]

[0431] Limit of Quantitative Analysis Limits of Quantitative Quantitative (LoQ) were determined in accordance with CLSI data collection guidelines.

[0432] The LoQ (Lowest Quantity) in an MTX assay is defined as the lowest concentration at which acceptable inter-assay precision and recovery are confirmed.

[0433] A series of samples were prepared by spiking negative pooled serum with MTX to theoretical concentrations of 0.020, 0.030, 0.040, and 0.050 μmol / L. Eight duplicate measurements of the test samples were performed: twice from the same calibration curve on day 1, twice from a recalibration curve on day 2, and once from a recalibration curve on day 3, for a total of 40 duplicate measurements. The results are summarized in Table 10. At 0.030 μmol / L, the accuracy was 4.87% CV, the standard deviation was 0.002, and the recovery rate was 113.6%, meeting the LoQ criteria.

[0434] Blank limit and detection limit The limit of blank (LoB) and the limit of detection (LoD) were evaluated by 60 repetitions of the blank (usually pooled human serum) and 60 repetitions of positive levels exceeding the LoB (approximately 0.020 μmol / L of MTX in serum). Three runs were performed, with 20 repetitions of the blank and 20 repetitions of low-concentration MTX samples in each run.

[0435] LoD refers to the actual concentration at which the observed test result is highly likely to exceed LoB, and therefore can be declared "detected."

[0436] [Table 10]

[0437] The following characteristics of the MTX assay were determined according to CLSI EP17-A2. LoQ was determined to be 0.030 μmol / L. LoB was calculated using a non-parametric method. The blank values ​​were sorted in ascending order, and the average of the 57th and 58th values ​​was calculated to obtain a LoB value of 0.000 μmol / L. LoD was determined by the following formula.

[0438] LoD = LoB + 1.652 (SDs)

[0439] [Table 11]

[0440] Specificity Specificity testing was performed using samples prepared by adding 0.050 μmol / L and 0.500 μmol / L of MTX and cross-reactants to negative pooled serum. Solvent controls were also prepared and tested for comparison.

[0441] Table 12a shows the interference rate (%) of compounds tested in the presence of MTX, and Table 12b shows the specificity in the absence of MTX.

[0442] [Table 12a]

[0443] [Table 12b]

[0444] Cross-reactivity with DAMPA was also tested. DAMPA stock solution was spiked into negative pooled serum in the absence of MTX, and the results are summarized in Table 12c.

[0445] [Table 12c]

[0446] Endogenous interfering substances The evaluation of endogenous interfering substances was performed in accordance with CLSI data collection guidelines. Pooled serum samples were used as control samples.

[0447] [Table 13]

[0448] The foregoing is merely illustrative of the principles of the embodiments of this disclosure. Those skilled in the art will understand that various configurations embodying the principles of the embodiments and falling within the spirit and scope of this disclosure can be devised, although these are not expressly described or illustrated herein. Furthermore, all examples and conditional statements described herein are intended primarily to help the reader understand the principles of the embodiments and the concepts to which the inventors have contributed to the development of this art, and should be interpreted as not being limited to the examples and conditions specifically described. Moreover, all descriptions relating to the principles, aspects, embodiments, and specific examples of this disclosure described herein are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include not only currently known equivalents but also equivalents to be developed in the future, i.e., all elements that perform the same function regardless of their structure.

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