WO2026030017A1 - Nitazene analogs and conjugates and methods of production and use thereof - Google Patents

Abstract

Haptens, conjugates, and immunogens for nitazene are provided herein. Also provided are kits including the haptens or conjugates and antibodies raised against the compounds as well as methods for detecting nitazene and/or a metabolite of nitazene in a sample.

Description

NITAZENE ANALOGS AND CONJUGATES AND METHODS OF PRODUCTION AND USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 63/676,960, filed on July 30, 2024. The entire disclosure of the above application is incorporated herein by reference. FIELD [0002] This disclosure generally relates to methods, compositions, and kits for detecting the presence and/or amounts of nitazene, analogs of nitazene, and/or metabolites of nitazene in biological samples. In particular, the disclosure relates to haptens, conjugates, and assays for detection of nitazene, its analogs, and/or its metabolites. BACKGROUND [0003] This section provides background information related to the present disclosure which is not necessarily prior art. [0004] The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. Over the last decade, testing for drugs of abuse has become commonplace. This testing is not only for the monitoring of criminal offenders and drug addicts, but employers also use it for the screening of workers. In recent years, immunoassays based on a reaction of an antibody with an antigen have been extensively investigated for this purpose. Immunoassays may be roughly classified into a radioimmunoassay using a radioactive isotope, an enzyme-immunoassay (EIA) using an enzyme, and a luminescence assay using fluorescent labels, e.g., fluorescence polarization, and chemiluminescent labels. [0005] Nitazene is a synthetic opioid containing a benzimidazole core. Nitazene is a selective μ-opioid agonist and was initially developed for its pain-relieving properties, but due to unacceptable side effects, such as respiratory depression, there is no acceptable medical use for nitazene. However, nitazene has begun to emerge as a drug of abuse in the narcotics markets. Therefore, an assay for detecting nitazene and/or a metabolite of nitazene in a biological sample is needed. SUMMARY [0006] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features. [0007] In certain aspects, the present disclosure provides a complex corresponding in structure to a Formula (I): wherein: ; hydrogen, an alkyl, an alkoxy, an ; X¹ is a halo; a, c, e, g, i, and k are each zero or 1; b, d, f, h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH₂CH_3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. [0008] In yet other aspects, the present disclosure provides a compound corresponding in structure to a Formula (Ia): (Ia) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , –O–, or –NH–; a a and e are each zero or 1; b and f are each zero to 100; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. [0009] In yet other aspects, the present disclosure provides a conjugate corresponding in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. [0010] In yet other aspects, the present disclosure provides a conjugate corresponding in structure to a Formula (Ic): wherein: R¹, R², and R³ are an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH₂CH₃ and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. [0011] In yet other aspects, the present disclosure provides a kit including an anti-nitazene antibody and a conjugate corresponding in structure to a Formula (Ib) as described herein or a Formula (Ic) as described herein. The anti-nitazene antibody may be raised against a complex as described herein wherein the immunogenic carrier is present. [0012] In yet other aspects, the present disclosure provides a method for detecting the presence of nitazene and/or a metabolite of nitazene in a sample. The method includes combining the sample, a conjugate, and an anti-nitazene antibody in a medium, and examining the medium for the presence of a complex comprising nitazene and the anti-nitazene antibody. The conjugate corresponds in structure to a Formula (Ib) as described herein or a Formula (Ic) as described herein. The anti-nitazene antibody may be raised against a complex as described herein wherein the immunogenic carrier is present. [0013] In yet other aspects, the present disclosure provides a method of preparing an antibody, the method including immunizing an antibody-producing animal with an immunogen comprising a composition comprising the complex as described herein, and isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen. [0014] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. DRAWINGS [0015] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure. [0016] FIG.1 is a reaction scheme 1 depicting an example of a synthesis of compound (I). [0017] FIG. 2 is a reaction scheme 2 depicting an example of a synthesis of conjugates and/or immunogens of compound (I). [0018] FIG.3 is a reaction scheme 3 depicting an example of a synthesis of intermediate compounds for synthesizing compounds (II) and (III). [0019] FIG.4 is a reaction scheme 4 depicting an example of a synthesis of intermediate compounds for synthesizing compound (II). [0020] FIG.5 is a reaction scheme 5 depicting an example of a synthesis of intermediate compounds for synthesizing compound (III). [0021] FIG. 6 is a reaction scheme 6 depicting an example of a synthesis of compound (II). [0022] FIG. 7 is a reaction scheme 7 depicting an example of a synthesis of compound (III). [0023] FIG. 8 is a reaction scheme 8 depicting an example of a synthesis of conjugates and/or immunogens of compound (II). [0024] FIG. 9 is a reaction scheme 9 depicting an example of a synthesis of conjugates and/or immunogens of compound (III). [0025] FIG.10 is a reaction scheme 10 depicting an example of a synthesis of compound (IV). [0026] FIG.11 is a reaction scheme 11 depicting an example of a synthesis of conjugates and/or immunogens of compound (IV). [0027] FIG.12 is a reaction scheme 12 depicting an example of a synthesis of compounds as described herein. [0028] FIG.13 is a reaction scheme 13 depicting an example of a synthesis of compounds and their corresponding conjugates and immunogens as described herein. [0029] FIG.14 is a reaction scheme 14 depicting an example of a synthesis of compounds and their corresponding conjugates and immunogens as described herein. [0030] FIG.15 is a reaction scheme 15 depicting an example of a synthesis of compounds and their corresponding conjugates and immunogens as described herein. DETAILED DESCRIPTION [0031] Example embodiments will now be described more fully with reference to the accompanying drawings. [0032] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. [0033] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. [0034] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses. [0035] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference. [0036] All of the articles, compositions, kits, and/or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, compositions, kits, and/or methods have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles, compositions, kits, and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims. A. Definitions [0037] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: [0038] The use of the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As such, the terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a compound” may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term “plurality” refers to “two or more.” [0039] The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example. [0040] The use of the term “or” in the claims is used to mean an inclusive “and/or” unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition “A or B” is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). [0041] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase “in some embodiments” or “one example” in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims. [0042] Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. [0043] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges. [0044] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for a composition/apparatus/ device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term “about” is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. [0045] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. Although the open- ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment. [0046] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated. [0047] It should be understood for any recitation of a method, composition, device, or system that “comprises” certain steps, ingredients, or features, that in certain alternative variations, it is also contemplated that such a method, composition, device, or system may also “consist essentially of” the enumerated steps, ingredients, or features, so that any other steps, ingredients, or features that would materially alter the basic and novel characteristics of the invention are excluded therefrom. [0048] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. [0049] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item. [0050] As used herein, the phrases "associated with" and “coupled to” include both direct association/binding of two moieties to one another as well as indirect association/binding of two moieties to one another. Non-limiting examples of associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non- covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example. [0051] The term “complex” encompasses large molecule and small molecule compounds and conjugates as described herein. [0052] The terms “analog” and “derivative” are used herein interchangeably and refer to a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also contain one or more substitutions thereto. The term “substitution” as used herein will be understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may include H, hydroxyl, thiol, a halide selected from fluoride, chloride, bromide, or iodide, a linear, branched or cyclic alkyl, optionally substituted, and linear branched or cyclic alkenyl, wherein the optional substituents are selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted wherein the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH (alkyl), -NH(cycloalkyl)₂, carboxy, and -C(O))-alkyl. [0053] The term "alkyl" refers to an organic group that consists solely of single-bonded carbon and hydrogen in either a straight, branched, or cyclic configuration. The number of carbon atoms in the organic group is 1 to 50, or 1 to 40, or 1 to 30, or 1 to 25, or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5, or 2 to 50, or 2 to 40, or 2 to 30, or 2 to 25, or 2 to 20, or 2 to 15, or 2 to 10, or 2 to 5, or 5 to 50, or 5 to 40, or 5 to 30, or 5 to 25, or 5 to 20, or 5 to 15, or 5 to 10. The term “lower alkyl” refers to alkyl wherein the number of carbon atoms in the organic group is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10. Examples of an alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, and so forth. Further, as used herein, “Me” refers to methyl, “Et” refers to ethyl, “Pr” refers to propyl, “i-Pr” refers to isopropyl, “Bu” refers to butyl, “t-Bu” refers to tert-butyl, “iBu” refers to isobutyl, “Pn” refers to pentyl, and “NPn” refers to neopentyl. [0054] The term “alkoxy” refers to an organic group of the number of carbon atoms designated above of either a straight, branched or cyclic configuration wherein the organic group includes an ether oxygen for linking an alkyl group as described herein to a parent compound. Examples of an alkoxy include methoxy (—OCH3), ethoxy (—OCH2CH3), propoxy (— OCH₂CH₂CH₃), and the so on. [0055] The term “halo” refers to fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I). [0056] The term “amine” or “amino” used alone or as part of a larger moiety refers to unsubstituted (—NH2). The term “alkylamine” refers to an alkyl substituted amine (—NR2), wherein each R group is a hydrogen or an alkyl substituent as described above, where at least one alkyl substituent is present. For example, the alkylamine may be mono-substituted (—NRH) or di-substituted (—NR₂) amine. Examples include methylamino (—NHCH₃), dimethylamino (— N(CH3)2), and so on. [0057] The term “nitazene” refers to the compound with the chemical structure shown below: . Nitazene also may be referred to as “2-(2-benzyl-5-nitrobenzimidazol-1-yl)-N,N- diethylethanamine.” [0058] The term “metabolite of nitazene” refers to a compound, such as an intermediate or a byproduct, formed by the metabolism of nitazene. Exemplary metabolites of nitazenes are shown below: [0059] The term “immunogen” refers to any substance capable of eliciting an immune response in an organism. [0060] The term “conjugate” refers to any substance formed from the joining together of two parts. Representative conjugates in accordance with the present invention include those formed by the joining together of a small molecule and a large molecule, such as a protein. The term “conjugate” subsumes the term “immunogen.” [0061] The term “hapten” refers to a portion of an immunogen that is typically low in molecular weight, which does not by itself stimulate antibody development. [0062] The phrase “activated hapten” refers to a hapten that has been provided with an available reaction site—for example, by the attachment of a linking group carrying a reactive moiety—that can be used to connect the hapten to a carrier, immunogen, label, tracer, or other moiety. [0063] The term “linking group” (or “linker”) refers to a chemical moiety that is used to connect a hapten to a macromolecular carrier, immunogen, label, tracer, or other moiety. The use of a linking group may or may not be advantageous or needed, depending on the specific hapten and carrier and desired specificity of antibody. Suitable linkers include straight, branched, saturated or unsaturated carbon chains, which may incorporate one or more heteroatoms—that is, atoms other than carbon (e.g., oxygen, nitrogen, sulfur, etc.)—within the chain or substituted onto and/or at a terminus thereof. [0064] The phrases “carrier” and “macromolecular carrier” refer to high molecular weight substances that can be coupled to haptens to form immunogens. Suitable macromolecular carriers include but are not limited to proteins, glycoproteins, polymers, polysaccharides, polypeptides, and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from a host. [0065] The term “polypeptide” refers to any compound formed by the linkage of two or more amino acids via an amide bond. Representative polypeptides include polymers of α-amino acids in which the α-amino group of each non-terminal amino acid residue is linked to the α- carboxyl group of an adjacent residue in a linear chain. High molecular weight polypeptides are referred to as “proteins.” [0066] The term “label” refers to a member of a signal producing system. The label is capable of being detected directly or is detectable through a specific binding reaction that produces a detectable signal. For example, a label may be an identifying tag that can be attached to a carrier substance or molecule to detect an analyte. The labels generally are radioisotopic, luminescent, particulate, or enzymic. The label can be a poly(amino acid), or protein, or non-poly(amino acid), isotopic or non-isotopic, usually non-isotopic, and can be a catalyst, such as an enzyme (e.g., β- galactosidase, peroxidase, etc.), a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule (e.g., rhodamine, fluorescein isothiocyanate or FITC, etc.), chemiluminescent molecule (e.g., dioxetanes, luciferin, etc.), coenzyme, enzyme substrate, radioactive group (e.g., ¹²⁵I), a protein-binding partner (e.g., biotin), a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0067] The term “non-poly(amino acid) labels” refers to those labels that are not proteins such as enzymes. A non-poly(amino acid) label may be a member of a signal producing system. The non-poly(amino acid) label is capable of being detected directly or is detectable through a specific binding reaction that produces a detectable signal. The non-poly(amino acid) labels generally are radioisotopic, luminescent, particulate, polynucleotidic, or the like. More particularly, the label can be isotopic or non-isotopic, usually non-isotopic, and can be a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescent molecule, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0068] The signal producing system may have one or more components, at least one component being the label. The signal producing system generates a signal that relates to the presence of an analyte in a sample. The signal producing system includes all of the reagents required to produce a measurable signal. Other components of the signal producing system may be included in a developer solution and can include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, and the like. Other components of the signal producing system may be coenzymes, substances that react with enzymic products, other enzymes and catalysts, and the like. The signal producing system provides a signal detectable by external means, by use of electromagnetic radiation, desirably by visual examination. Exemplary signal-producing systems are described in U.S. Pat. No.5,508,178 (Rose, et al.), the relevant disclosure of which is incorporated herein by reference. [0069] The term “immunogenic carrier” refers to a group which, when conjugated to a hapten and injected into a mammal, will induce an immune response and elicit the production of antibodies that bind to the hapten. Haptens are compounds capable of binding specifically to corresponding antibodies, but do not themselves act as immunogens (or antigens) for preparation of the antibodies. Antibodies that recognize a hapten can be prepared against compounds comprised of the hapten linked to an immunogenic (or antigenic) carrier. Immunogenic carriers are also referred to as antigenic carriers. Typical immunogenic carriers include, without limitation, poly(amino acids), polysaccharides, nucleic acids, and particles (biologic and synthetic materials). A wide variety of such carriers are disclosed in Davalian, et al., U.S. Pat. No.5,089,390, column 4, line 57 to column 5, line 5, incorporated herein by reference. Immunogenic carriers include proteins such as, for example, albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, and so forth. Illustrative proteins include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin (OVA), bovine gamma-globulin (BGG), bovine thyroglobulin (BTG), glucose-6-phoshpate dehydrogenase (G6PDH), and the like. [0070] The term “sample” as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like. [0071] The term “specific binding partner” or “analyte-specific binder” will be understood to refer to any molecule capable of specifically associating with a target analyte. For example, but not by way of limitation, the binder/binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), any fragments thereof, and any combinations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte. [0072] The term “antibody” is used in the broadest sense, and specifically (but not by way of limitation) covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired biological activity of analyte binding. Thus, the term “antibody” or “antibody peptide(s)” refers to a full- length immunoglobulin molecule (i.e., an intact antibody) or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins/peptides), and combinations or derivatives thereof. See, e.g., Hudson et al. (Nature Med. (2003) 9:129-134). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). [0073] The term “antigen binding fragment” or “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments are obtained using conventional recombinant and/or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies. [0074] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. [0075] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes. [0076] The terms “CDR,” and its plural “CDRs,” refer to a complementarity determining region (CDR) of an antibody or antibody fragment, which determine the binding character of an antibody or antibody fragment. In most instances, three CDRs are present in a light chain variable region (CDRL1, CDRL2 and CDRL3) and three CDRs are present in a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. Among the various CDRs, the CDR3 sequences, and particularly CDRH3, are the most diverse and therefore have the strongest contribution to antibody specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety). [0077] The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three- dimensional structural characteristics (e.g., a “conformational epitope”), as well as specific charge characteristics. [0078] An epitope is defined as “the same” as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise epitopes that are the same. In certain embodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope. [0079] An antibody “specifically binds” an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules. In certain embodiments, an antibody comprises an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, the antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope or closely related epitopes. In certain instances, for example, homologous proteins from different species may comprise the same epitope. In certain embodiments, an antibody specifically binds to an antigen with a dissociation constant of no greater than 10^-6 M, 10^-7 M, 10^-8 M or 10^-9 M. When an antibody specifically binds to a receptor or ligand (i.e., counterreceptor), it may substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when an excess of antibody reduces the quantity of receptor bound to ligand by at least about 20%, 40%, 60% or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay). [0080] An “isolated” antibody is one which has been separated and/or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In certain embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, alternatively, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the environment in which the antibody is produced will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In addition, the “isolated antibody” is substantially free of other antibodies having different antigenic specificities. An isolated antibody may, however, have some cross-reactivity to other, related antigens. [0081] The term “antibody mutant” refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, such as at least 80%, or at least 85%, or at least 90%, or at least 95%. [0082] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that in one method of production they may be synthesized by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, in one embodiment, the monoclonal antibodies produced in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)). [0083] The monoclonal antibodies utilized in accordance with the present disclosure may be produced by any methodology known in the art including, but not limited to, a result of a deliberate immunization protocol; a result of an immune response that results in the production of antibodies naturally in the course of a disease or cancer; phage-derived antibodies; and the like. In addition to the hybridoma production method listed above, the monoclonal antibodies of the present disclosure may be produced by other various methods such as, but not limited to, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567); isolation of antibody fragments from a phage display library (see, e.g., Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); as well as various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)). Further, many monoclonal antibodies that may be utilized in the conjugates and methods disclosed or otherwise contemplated herein are widely commercially available, and therefore no further description thereof is deemed necessary. [0084] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Generally, a substantially pure composition will comprise more than about 50% percent of all macromolecular species present in the composition, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. [0085] An “analyte” is a molecule that is capable of being recognized by an analyte- specific binding partner, such as (but not limited to) an antibody. An analyte comprises at least one antigenic determinant or "epitope," which is the region of the analyte which binds to the analyte-specific binding partner (i.e., antibody). As used herein, the term “analyte” subsumes the term “antigen,” which refers to any compound that can bind to an antibody. Furthermore, as used herein, the term “analyte” refers to all manner of chemical substances including but not limited to: conjugates; immunogens; drugs; drug metabolites; drug derivatives; hormones; proteins; antigens; oligonucleotides; and the like. [0086] The phrase “detecting an analyte” refers to any quantitative, semi-quantitative, or qualitative method, as well as to all other methods for determining an analyte in general. For example, a method that merely detects the presence or absence of nitazene, an analog of nitazene, and/or a metabolite of nitazene in a sample lies within the scope of the present disclosure, as do methods that provide data as to the amount or concentration of the drug in the sample. The terms “detecting,” “determining,” “identifying,” and the like are used synonymously herein, and all lie within the scope of the present invention. [0087] The phrase “reagent kit” or term “kit” refers to an assembly of materials that are used in performing an assay. The reagents can be provided in packaged combination in the same or in separate containers, depending on their cross-reactivities and stabilities, and in liquid or in lyophilized form. The amounts and proportions of reagents provided in the kit can be selected so as to provide optimum results for a particular application. A reagent kit embodying features of the present invention comprises antibodies specific for nitazene, metabolites of nitazene, analogs of nitazene, conjugates of nitazene analogs, and/or enzymes or proteins necessary for detecting the presence and quantity of the antibody, nitazene drug, and/or metabolite of nitazene in a sample. The kit may further comprise calibration and control materials. The reagents may remain in liquid form or may be lyophilized. [0088] The phrase “calibration and control materials” refers to any standard or reference material containing a known amount of an analyte to be measured. A sample suspected of containing an analyte and the corresponding calibration material are assayed under similar conditions. The concentration of analyte is calculated by comparing the results obtained for the unknown specimen with the results obtained for the standard. This is commonly done by constructing a calibration curve. [0089] The terms “identical,” “sequence identity,” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window. The degree of amino acid or nucleic acid sequence identity for purposes of the present disclosure is determined using the BLAST algorithm, described in Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403–10, which is incorporated herein by reference. The BLAST algorithm is publicly available through software provided by the National Center for Biotechnology Information (at the web address www.ncbi.nlm.nih.gov). [0090] Various additional R groups, conjugates, systems, kits, and methods that may be utilized in combination with the compositions of the present disclosure and/or in methods of detecting nitazene are disclosed in US Patent No. 7,022,492 (the entirety of which is hereby expressly incorporated herein by reference). [0091] Thus, in accordance with the present disclosure, there have been provided compositions, kits, and devices, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure. B. Nitazene Analogs, Immunogens, and Conjugates [0092] Compounds, such as nitazene analogs, which can be used for preparing immunogens, conjugates, and antibodies useful in immunoassays for the determination of nitazene and/or a metabolite of nitazene, are provided herein. Immunogens and conjugates formed from the compounds are also provided herein. It is contemplated herein that nitazene analogs include haptens and activated haptens. [0093] In any embodiment, such nitazene, immunogen, and/or conjugate may be a complex corresponding in structure to a Formula (I): wherein: ; hydrogen, an alkyl, an alkoxy, an ; a a, c, e, g, i, and k are each zero or 1; b, d, f, h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label. [0094] In any embodiment, when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. For example, a complex corresponding in structure to Formula (I) may not be one or more of the following compounds: , , , , , , , , , _{or .} [0095] In any embodiment, L¹ may be ; R² and R³ each independently may be hydrogen, an alkyl, an alkoxy, an alkylamine, , , , , , or ; R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ each independently may be –CH2–, –O–, or –NH–; X¹ may be a halo; a, c, e, g, i, and k each may be zero or 1; b, d, f, h, j, and l each may be zero to 100; and each of Y¹, Y², and Y³ may be an immunogenic carrier or a label. [0096] In any embodiment, L¹ may ; R¹, R², and R³ each independently may be hydrogen, an alkyl, an alkoxy, an , or ; R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ each independently may be –CH₂–, a halo; a, c, e, g, i, and k each may be zero or 1; b, d, f, h, j, and l each may be zero to 100; and each of Y¹, Y², and Y³ may be an immunogenic carrier or a label. [0097] In any embodiment, the alkyl may be C1-C20-alkyl, C1-C12-alkyl, C1-C10-alkyl, C1- C₈-alkyl, C₁-C₆-alkyl, C₁-C₄-alkyl, C₁-C₃-alkyl, C₁-C₂-alkyl, or methyl. For example, the alkyl may be C1-C6-alkyl, C1-C4-alkyl, C1-C3-alkyl, C1-C2-alkyl, or methyl. [0098] In any embodiment, the alkoxy may be C₁-C₁₂-alkoxy, C₁-C₁₀-alkoxy, C₁-C₈- alkoxy, C1-C6-alkoxy, C1-C4-alkoxy, C1-C3-alkoxy, C1-C2-alkoxy, or methoxy. For example, the alkoxy may be C₁-C₆-alkoxy, C₁-C₄-alkoxy, C₁-C₃-alkoxy, C₁-C₂-alkyl, –OCH₃, –OCH₂CH_3, – O(CH2)2CH3, or –OCH(CH3)2. [0099] In any embodiment, the alkylamine may be —NR₂, wherein each R group is a hydrogen or an alkyl substituent as described above, where at least one alkyl substituent is present. For example, the alkyl be a C1-C12-alkyl, C1-C10-alkyl, C1-C8-alkyl, C1-C6-alkyl, C1-C4-alkyl, C1- C₃-alkyl, C₁-C₂-alkyl, or methyl. Exemplary alkylamines include, but are not limited to, methylamino (—NHCH3), dimethylamino (—N(CH3)2), or ethylmethylamino (—N(C₂H₅)(CH₃)). [0100] Additionally, each halo may be fluoro (F), chloro (Cl), bromo (Br), or iodo (I). For example, each halo may be Cl or Br. [0101] Additionally, b, d, f, h, j, and l each independently may be zero to 75, zero to 50, zero to 40, zero to 30, zero to 25, zero to 20, zero to 15, zero to 10, zero to 5, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5. For example, b, d, f, h, j, and l each may be 1 to 25, 1 to 10, or 1 to 5. [0102] Suitable immunogenic carriers include, but are not limited to, a protein, a polypeptide, a polysaccharide, a nucleic acid, and a particle (e.g., biologic and synthetic materials). A wide variety of such carriers are disclosed U.S. Pat. No. 5,089,390, incorporated herein by reference. Examples of suitable proteins include, but are not limited to, albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, and so forth. Nonlimiting examples of proteins include the keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6- phoshpate dehydrogenase (G6PDH). A protein may be attached to a linking group by means of an amine group on the protein. [0103] In any embodiment, a label may be radioisotopic, luminescent, particulate or enzymic. The label can be a poly(amino acid), or protein, or non-poly(amino acid), isotopic or non-isotopic, and can be a catalyst, such as an enzyme (e.g., β-galactosidase, peroxidase, etc.), a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule (e.g., rhodamine, fluorescein isothiocyanate or FITC, etc.), chemiluminescent molecule (e.g., dioxetanes, luciferin, etc.), coenzyme, enzyme substrate, radioactive group (e.g., ¹²⁵I), a protein-binding partner, biotin or another small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0104] In any embodiment, R¹ may be hydrogen, an , _{[0105] In any embodiment, R2 may be hydrogen, ,} or [0106] In any embodiment, may R³ may be hydrogen, an , be hydrogen, and R³ R⁴, R⁵, and R⁶ each may may be zero or 1, and b, d, and f each may be 1 to 25, 1 to 10, or 1 to 5. For example, R¹ may be C1-C2 alkyl, such as methyl, R² may be hydrogen, R³ may , R⁴ may be – O–, a may be 1, and b may be 1 to 10 or 1 to 5, such as 1, 2, R¹ may be C1-C2 alkyl, such as methyl, R² may be hydrogen, R³ R⁵ may be –O– , c may be 1, and d may be 1 to 10 or 1 to 5, such as 1, R¹ may be C₁- C₂ alkyl, such as methyl, R² may be hydrogen, R³ may , R⁶ may be – O–, X¹ may be Br, e may be 1, and f may be 1 to 10 or 1 [0108] In any embodiment, R¹ may be , , or , R² may be hydrogen, and R³ may be hydrogen, C₁-C₆ alkoxy, C₁-C₄ R⁴, R⁵, and R⁶ each may be –CH2–, –O–, or –NH–, X¹ may be halo, as or a, c, e each may be zero or 1, and b, d, and f each may be 1 to 25, 1 to 10, or 1 _{to 5. For example, R1 , R2 may be hydrogen, R3 may be hydrogen,} –OCH3, –OCH2CH3, –O 2, R⁴ may be –CH2–, a may be 1, and b may be 1 to 10 or 1 to 5, such as or a may be zero. In another example, R¹ may m_{ay be hydrogen, R3 may be hydrogen, –OCH3, –OCH2CH3, –O(CH2)2-} may be –CH₂–, c may be 1, and d may be 1 to 10 or 1 to 5, such as 1, 2, or 3. Alternatively, c may be zero. In another example, R¹ may , R² may be hydrogen, R³ may be hydrogen, –OCH3, –OCH2CH3, ⁶ 2, R may be –CH₂–, X¹ may be Br, e may be 1, and f may be 1 to 10 or 1 to 5, such as 1, 2, or 3. Alternatively, e may be zero. [0109] In any embodiment, R¹ may be C₁-C₆ alkyl, C₁-C₄ alkyl, or C₁-C₂ alkyl, R² may be C1- –NH–, X¹ may be halo, such as Cl or Br, a, c, and e may be zero or 1, and b, d, and f may be 1 to 25, 1 to 10, or 1 to 5. For example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be may be hydrogen, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, or –OCH(CH₃)₂, be 1, and b may be 1 to 10 or 1 to 5, such as 1, 2, or 3. Alternatively, a may be zero. In another example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be , R³ may be hydrogen, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, or –OCH(CH₃)₂, R⁵ may be –CH2–, c may be 1, and d may be 1 to 10 or 1 to 5, such as 1, 2, or 3. Alternatively, c may be zero. In another example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be , R³ may be hydrogen, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, or – –CH2–, X¹ may be Br, e may be 1, and f may be 1 to 10 or 1 to 5, such as or e may be zero. [0110] In any embodiment, the alkyl may be a C1-C4 alkyl, such as a C1-C2 alkyl, for example, methyl; the alkoxy may be a C₁-C₄ alkoxy, such as a C₁-C₂ alkoxy, for example, methoxy; each halo may be Cl or Br, for example Br; b, d, f, h, j, and l each may be 1 to 25, such as 1 to 10, for example, 1 to 5; and the immunogenic carrier may be selected from the group consisting of a protein, a polypeptide, and a polysaccharide. [0111] In any embodiment, the complex may correspond in structure to any one of Formulas (A)-(S): , , , , , , N N H N N Br N m O O₂N N NO₂ N O R³ _{O NH Formula (I)} n _{Y1 , Formula (J) ,} , , O , R² may be hydrogen, R³ may be hydrogen, –OCH3, –OCH2CH3, –O(CH2)2CH3, or – OCH(CH₃)₂; m and n each may be zero to 100, such as 1 to 25, for example, 1 to 10; and each of Y¹, Y², and Y³ may be an immunogenic carrier or a label. The immunogenic carrier may be a protein, a polypeptide, or a polysaccharide, wherein the protein may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [0112] In a further embodiment, provided herein are nitazene analog or hapten compounds encompassed by Formula (I), which correspond in structure to a Formula (Ia): wherein: 1 ² R , R , and R³ are an alkyl, an alkoxy, , –O–, or –NH–; X¹ is a halo; a and e are each zero or 1; and b and f are each zero to 100. [0113] In any embodiment, when R¹ is hydrogen or –CH₂CH_3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. For example, a compound corresponding in structure to Formula (Ia) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0114] In any embodiment, the alkyl may be a C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₂ alkyl, or methyl. The alkoxy may be a C1-C6 alkoxy, C1-C4 alkoxy, a C1-C2 alkoxy or methoxy. Each halo may Cl or Br, or Br, and b and f each may be 1 to 25, 1 to 10, or 1 to 5. [0115] In any embodiment, R¹ may be C1-C6 alkyl, such as a C1-C4 alkyl, C1-C2 alkyl, or methyl or R¹ . [0116] In be hydrogen. [0117] In any embodiment, R³ may be or . Alternatively, R³ may be hydrogen or a C1-C6 alkoxy, C1-C4 alkoxy, a C1-C2 alkoxy or methoxy. [0118] In any embodiment, R¹ may be C₁-C₆ alkyl, C₁-C₄ alkyl, or C₁-C₂ alkyl, R² may be hydrogen, and R³ may be . R⁴ may be –CH₂–, –O–, or –NH–, a may be zero or 1, and b may be 1 to For example, R¹ may be C1-C2 alkyl, such as methyl, R² may be hydrogen, R³ may , R⁴ may be –O–, a may be 1, and b may be 1 to 5, such as 1, 2, or 3. [_{0119] In a further embodiment, R1 , R4 may be –CH2–, –O–} , or –NH–, a may be zero or 1, b may be 1 to R² may be hydrogen, and R³ may be hydrogen or a C₁-C₆ alkoxy, or a C₁-C₄ alkoxy, or a C₁-C₂ alkoxy. For example, R¹ may , _{a may be zero, b may be 1 to 5, such as 1, 2, or 3, R2 may be hydrogen,} Alternatively, a may be 1 and R⁴ may be –CH₂–. In another example, R_{1 , a may be zero, b may be 1 to 5, such as 1, 2, or 3, R2 may be} C alkoxy, ⁴ 2 such as methoxy. Alternatively, a may be 1 and R may be –CH₂–. [0120] In a further embodiment, R¹ may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R² may be hydrogen, R³ may , R⁶ may be –CH2–, –O–, or –NH–, e may be zero or 1, f may be 1 to X¹ may be Cl or Br. For example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be hydrogen, R³ may , R⁶ may be –O–, e may be 1, f may be 1 to 5, such as 1, 2, or 3, and X¹ [0121] Nonlimiting examples of compounds corresponding in structure to Formulas (I) and (Ia) are shown below in Table 1. Table 1 [ ] n a ur er emo men, prov e eren are conjugaes encompasse y ormula (I), which correspond in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label. [0123] In any embodiment, when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. For example, a compound corresponding in structure to Formula (Ib) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0124] In any embodiment, R¹ may be hydrogen, an , , [0126] In any embodiment, may R³ may be hydrogen, an . C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or methyl, R² may be hydrogen, R³ or each may be –CH2–, –O–, or –NH–, g, i, and k each 1 to 25, 1 to 10, or 1 to 5, and each of Y¹, Y², and Y³ may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). For example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be hydrogen, R³ may , R⁷ may be –O–, g may be 1, and h may be 1 to 10 or 1 to 5. In C₂ alkyl, such as methyl, R² may be hydrogen, R³ m_{ay be –O–, i may be 1, and j may be 1 to 10 or 1 to 5. In} C₂ alkyl, such as methyl, R² may be hydrogen, R³ may be C₄ zero or 1, h, j, and l each may be 1 to 25, 1 to 10, or 1 to 5, and each of Y¹, Y², and Y³ may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (_{G6PDH). For example, R1 , R2 may be hydrogen, R3 may be} hydrogen, –OCH3, –OCH2CH3, 2, R⁷ may be –CH2–, g may be 1, and h may be 1 to 10 or 1 to 5. Alternatively, g may be zero. In another example, R¹ may be , R² may be hydrogen, R³ may be hydrogen, –OCH₃, –OCH₂CH_3, – (CH3)2, R⁸ may be –CH2–, i may be 1, and j may be 1 to 10 or 1 to 5. Alternatively, i may be zero. In another example, R¹ may , R² may be hydrogen, R³ may be hydrogen, –OCH3, –OCH2CH3, –O(CH2)2CH3, or –OCH(CH3)2, R⁹ may be –CH₂–, k may be 1, and l may be 1 to 10 or 1 to 5. Alternatively, k may be zero. [0129] In any embodiment, R¹ may be may be C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or m_{ethyl, R2 may , or} , R³ may be may be hydrogen, C₁-C₆ alkoxy, C₁-C₄ alkoxy, or a C₁- each may be –CH2–, –O–, or –NH–, g, i, and k are each zero or 1, h, j, and l each may be 1 to 25, 1 to 10, or 1 to 5, and each of Y¹, Y², and Y³ may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). For e_{xample, R1 may be C1-C2 alkyl, such as methyl, R2 , R3 may be} hydrogen, –OCH3, –OCH2CH3, –O(CH2)2CH3, or may be 1, and h may be 1 to 10 or 1 to 5. Alternatively, g may be zero. In another example, R¹ may be C₁- C_{2 alkyl, such as methyl, R2 R3 may be hydrogen, –OCH3, –} OCH2CH3, –O(CH2)2CH3, or be 1, and j may be 1 to 10 or 1 to 5. Alternatively, i may be zero. In another example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be hydrogen, –OCH3, –OCH2CH3, –O(CH2)2CH3, or be 1, and l may be 1 to 10 or 1 to 5. Alternatively, k may be zero. [0130] In a further embodiment, provided herein are conjugates encompassed by Formula (I), which correspond in structure to a Formula (Ic): (Ic) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g are zero or h and l are each zero to 100; and each of Y¹ and Y³ is an immunogenic carrier or a label. [0131] In any embodiment, when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. For example, a compound corresponding in structure to Formula (Ic) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0132] In any embodiment, R¹ may be C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₂ alkyl, or methyl or R¹ . R² may be hydrogen. [_{0134] In any embodiment, R3 may be ,} , C1-C6 alkoxy, C1-C4 alkoxy, C1-C2 alkoxy, or methoxy. R⁷ and R⁹ each independently may be –CH₂– or –O–, h and l each may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, and a polysaccharide. [0136] In any embodiment, R¹ may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R² may be hydrogen, R³ may , R⁷ may be –O– or –CH₂–, g may be zero or 1, h may be 1 to 25, 1 to carrier may be a protein, a polypeptide, or a polysaccharide. C₁-C₂ alkyl, such as methyl, R² may be hydrogen, R³ may be –O–, g may be 1, h may be 1 to 5, such as 1, 2, or 3, and (KLH), bovine serum albumin (BSA), bovine , (OVA), bovine gamma globulin (BGG), or glucose-6- phosphate dehydrogenase (G6PDH). [0137] In various aspects, a conjugate may correspond in structure to: , where Y¹ may be keyhole limpet hemocyanin thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [_{0138] In any embodiment, R1 , R2 may be hydrogen, R3} may be hydrogen or C1-4 alkoxy, R⁷ may zero or 1, h may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, or a p_{olysaccharide. For example, R1 , R2 may be hydrogen, R3 may} be C1-2 alkoxy, such as methoxy, g 5, such as 1, 2, or 3, and Y¹ may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). Alternatively, g may be 1 and R⁷ may be –CH₂–. In another example, _{R1 be , R2 may be hydrogen, R3 may be hydrogen, g may be zero, h} may Y¹ may be keyhole limpet hemocyanin (KLH), bovine serum , (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). Alternatively, g may be 1 and R⁷ may be –CH2–. [0139] In various aspects, a conjugate may correspond in structure to: Y¹ , egg , gamma , or dehydrogenase (G6PDH). [0140] In any embodiment, R¹ may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R² may be hydrogen, R³ may be –CH₂–, –O–, or –NH–, k may be zero or 1,l may immunogenic carrier may be a protein, a polypeptide, or a polysaccharide. For example, R¹ may be C₁-C₂ alkyl, such as methyl, R² may be hydrogen, R³ may be –O–, k may be 1, l may be 1 to 5, such as 1, 2, or 3, (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH).

[0141] In various aspects, a conjugate may correspond in structure to: , where Y³ may be keyhole limpet hemocyanin (KLH), thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma , or phosphate dehydrogenase (G6PDH). [0142] In any embodiment, a conjugate may correspond in structure to: , hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH). C. Synthesis [0143] The syntheses of representative examples of the above compounds (e.g., nitazene analogs, haptens, activated haptens, immunogens, and conjugates) are discussed herein by way of illustration and not limitation. Other synthetic procedures will be suggested to those skilled in the art in view of the disclosure herein. Other compounds within the scope of the present invention may be prepared using suitable variants of the reagents employed below. The reaction temperatures and time are those customary for the type of reactions conducted and should be evident to those skilled in the art. [0144] For immunogen and conjugate syntheses, a protein may be attached to a linking group by means of an amine group or a thiol group on the protein. The formulas and compounds described herein may show the nitrogen atom of the amine group of the protein or the sulfur atom of the thiol group of the protein. In general, functional groups suitable for attaching a compound as described herein (e.g., hapten, activated hapten) to an immunogenic carrier (e.g., protein, enzyme) or label are usually an activated ester or alkylating agent when the amino acid(s) that are to be conjugated on the enzyme have amino or hydroxyl groups and are usually alkylating agents or the like when the amino acid(s) that are to be conjugated on the immunogenic carrier comprise a sulfur atom such as, e.g., a cysteine. A large number of suitable functional groups are available for attaching to amino groups and alcohols such as activated esters including imidic esters, sulfonic esters and phosphate esters, activated nitrites, aldehydes, ketones, alkylating agents and the like. Conjugation of haptens to proteins using these and other attaching groups are well known in the art and are described in reviews such as for example, Maggio, E. T. “Enzyme- Immunoassay” (CRC Press, Boca Raton, Fla., 1980), Chapter 4, which contains an assortment of conjugation techniques; pages 81–88 of which are incorporated herein by reference. [0145] Following reaction of the immunogenic carrier (e.g., protein, enzyme) with a compound such as discussed above to form a conjugate, the product is then optionally purified as may be required. The purification and characterization of poly(amino acid)-hapten conjugates has been described in detail by Maggio, et al.; “Enzyme-immunoassay” (CRC Press, Boca Raton, Fla., 1980), Chapter 4, pages 86–88 of which are incorporated herein by reference. For example, the protein-hapten conjugate can be purified, for example, by dialysis against aqueous/organic and aqueous solutions or by gel filtration chromatography on a support such as Sephadex®, and the like. [0146] As mentioned above, the conjugation can involve binding of a hapten to a free thiol group present on an amino acid side chain of the enzyme (e.g. cysteine). Such conjugation involves alkylation of the thiol sulfur atom by treatment with an electrophilic compound such as an alpha- or beta-unsaturated amide, ketone, ester, or the like, or an alkylating agent such as a reactive halide, e.g., bromide, or sulfonate or the like or reaction with an active disulfide such as a 2-nitro-4-carboxyphenyl disulfide. Specific examples by way of illustration and not limitation include alpha-bromoamides, maleimides, vinyl sulfones, alpha-iodoketones, and the like. [0147] Conjugation reactions with proteins or enzymes can be affected by a number of factors. These include, but are not confined to, pH, temperature, buffer, ionic strength, substances which may protect the enzyme active site, amount and type of cosolvent, reaction time, and activation chemistry. A range of pH values from about 5.0 to about 9.5 can usually be used for conjugation reactions. These reactions are generally carried out at about 0ºC to about 40ºC, preferably about 4ºC to about 20ºC. [0148] A number of buffers and salts, both alone and in combination, can be used for such reactions. These include Tris, bicarbonate, phosphate, pyrophosphate, ethylenediaminetetraacetic acid (EDTA), KCl, NaCl, and many others. The active site may be protected by substrates (i.e., glucose-6-phosphate and compounds that react reversibly with lysine (i.e., pyridoxal) to reduce deactivation of the enzyme during conjugation. [0149] Co-solvents which may enhance hapten solubility include, but are not limited to, dimethylformamide, carbitol, dimethyl sulfoxide, 1-methyl-2-pyrrolidinone, and 1,3-dimethyl- 3,4,5,6-tetrahydro 2(1H)-pyrimidinone. These may be useful as about 1 to about 30% of the reaction volume. Reactions can vary from about 15 min to many days, depending on the activation chemistry. Carboxylic compounds may be activated to form esters with N-hydroxysuccinimide or its sulfo-analog, or to mixed anhydrides through reaction with carbitol chloroformate or t- butylchloroformate, or may be coupled directly using carbodiimides such as EDC. For reaction with cysteine thiols on the enzyme, the hapten should contain a good leaving group such as I, Br, or tosyl; alternatively, the hapten can contain a thiol, preferably activated with 2,2′ dithiodipyridine, 5,5′dithiobis(2-nitrobenzoic acid) (DTNB), dithioerythritol (DTE), and the like. [0150] Another method of conjugation, described in Rowley, G. L., D. Leung, and P. Singh (U.S. Pat. No. 4,220,722) involves modification of the immunogenic carrier (e.g., protein, enzyme) with bromoacetyl containing reactants; the bromo groups are subsequently reacted with thiol-containing haptens. The reaction of the immunogenic carrier (e.g., protein, enzyme) with bromoacetyl modifier, and the bromoacetyl enzyme with the thiolated hapten, are subject to the same reaction condition variables described above. [0151] Referring to FIG. 1, synthesis of compound (I) may include combining 2-(4- hydroxyphenyl)acetic acid (compound 1) and imidazole in a suitable solvent (e.g., dichloromethane (DCM)), and then tert-butyldimethylsilyl chloride (TBDMSCl) may be added in a suitable amount. The organic layer may be washed with deionized (DI) water and then evaporated to dryness to yield a crude product including a di-TBDMS-derivative. The crude product may be dissolved in a suitable solvent (e.g., tetrahydrofuran (THF)) and washed with Na2CO3 aqueous solution for a suitable amount of time (e.g., about 30 mins). The organic solvent may be removed (e.g., on a rotatory evaporator), a suitable acid (e.g., hydrochloric acid (HCl)) may be added to achieve a suitable pH (e.g., pH~4), then ethyl acetate (EtOAc) may be used to extract compound 2 (TBDMSO-Ph-COOH). Compound 2 may be purified via liquid chromatography (LC). Compound 2 (TBDMSO-Ph-COOH) may be dissolved in a suitable solvent (e.g., DCM) and N,N-dimethylformamide (DMF) and oxalyl chloride may be added. The resulting reaction mixture may be blanketed with an inert gas (e.g., argon). The volatiles may be removed (e.g., on a rotatory evaporator) and the resulting oil may be further dried (e.g., on an oil pump) to yield compound 3 (TBDMSO-Ph-COCl). Compound 3 and a suitable solvent (e.g., THF) may be combined to form TBDMS-Ph-COCl-THF solution. A suitable solvent (e.g., THF), 2- fluoro-5-nitroaniline, and NEt₃ may be combined to form a reaction mixture. The resulting reaction mixture may be combined with the TBDMS-Ph-COCl-THF solution (e.g., added dropwise) and blanketed with an inert gas (e.g., argon) to yield a yellow solid. The yellow solid may be filtered off, and the filtrate may be mixed with silica gel and dried (e.g., on a rotatory evaporator), and furhter purified by LC to yield fractions containing compound 4, which may be further concentrated. Compound 4 may be combined with a suitable solvent (e.g., dimethylacetamide (DMAc)), N,N-diethyl-ethylenediamine and N,N-diisopropylethylamine (DIPEA). The resulting reaction mixture may be blanketed with an inert gas (e.g., argon). The reaction mixture may be heated at a suitable temperature (e.g., 70-100°C) for a suitable amount of time (e.g., about 3 days) to yield compound 6. DMAc may be evaporated (e.g., on a rotatory evaporator), then EtOAc may be added and washed with brine and DI water. The solvent may be removed (e.g., in vacuum) to yield nitazene-4-OH (compound 6) as a yellow solid. Nitazene-4- OH (compound 6) may be dissolved in a suitable solvent (e.g., DMF) and combined with K2CO3 and tert-butyl-bromo valerate. The resulting reaction mixture may be filtered off and the precipitate can be further extracted with EtOAc. The combined organic layers may be concentrated (e.g., on a rotatory evaporator) to give a yellow oil, which may be dissolved in acetonitrile (ACN) and purified (e.g., by high performance liquid chromatography (HPLC)) to yield fractions containing compound 7, which may be further lyophilized. [0152] Still referring to FIG.1, Compound 7 may be suspended in a solvent (e.g., DCM) and trifluoroacetic acid (TFA). The volatiles may be removed (e.g., in vacuo) from the suspension to yield Nitazene-O4-C5-OH (compound (I)). [0153] Referring to FIG. 2, Nitazene-O4-C5-OH (compound (I)) may be activated by suspending Nitazene-O4-C5-OH (compound (I)) in a solvent (e.g., DMF) and adding N-(3- dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC*HCl) and N- hydroxysuccinimide (SuOH) to give activated Nitazene-O4-C5-OSu (compound 9). Activated Nitazene-O4-C5-OSu (compound 9) may be dissolved in a solvent (e.g., DMF) to produce a Nitazene-O4-C5-OSu-DMF solution. Each protein such as, for example, OVA, BSA, KLH, and the like may be suspended in a suitable buffer and combined with the Nitazene-O4-C5-OSu-DMF solution to produce NitazeneO4-C5-OVA conjugate (compound 10a), NitazeneO4-C5-BSA conjugate (compound 10b), and NitazeneO4-C5-KLH immunogen (compound 10c). Reaction conditions include, for example, a buffer solution at pH of about 7 to 9, about 7.5 to 8.5, about 8. Such buffer solutions include, for example, phosphate or borate buffer etc., and combinations thereof. The resulting immunogens and conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer (PB), etc. The attachment of a protein to the linking moiety of the molecule can be through the amino group on a protein, where the nitrogen of the amino group may be the nitrogen of the linking group depicted above. [0154] Still referring to FIG.2, to prepare a G6PDH conjugate with compound (I), G6PDH enzyme may be buffer exchanged with a buffer (e.g., PB) and can be further diluted with the buffer. Glucose-6-phosphate di-sodium salt (G6PDNa2) and nicotinamide adenine dinucleotide (β-NADH) may be added to the enzyme solution. Additional solvent (e.g., DMF) may optionally be added to the enzyme solution. The enzyme solution may be combined with Nitazene-O4-C5- OSu-DMF solution, for example, in various concentrations, to produce NitazeneO4-C5-G6PDH 5x (compound 10d), NitazeneO4-C5-G6PDH 10x (compound 10e), NitazeneO4-C5-G6PDH 15x (compound 10f), NitazeneO4-C5-G6PDH 20x (compound 10g), and NitazeneO4-C5-G6PDH 25x (compound 10h). The resulting conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [0155] Referring to FIG. 3, to prepare Nitazene-N-C5-COOH and MeO-Nitazene-N-C5- COOH haptens (compounds (II) and (III)), K₂CO₃, 1-(Boc-amino)-2-(ethylamino)ethane solid (compound 11), and tert-Butyl 5-bromovalerate (compound 12) in acetonitrile (ACN) may be combined and heated to a suitable temperature (e.g., 80°C ) for a suitable amount of time (e.g., about 20 hours). The solid may be filtered from the reaction mixture and washed with ACN, and the combined acetonitrile filtrate may be concentrated (e.g., on a rotatory evaporator) to afford a colorless solid as compound 13. The solid (compound 13) may be treated with TFA-DCM for a suitable amount of time (e.g., about 8-12 hours) at a suitable temperature (e.g., about 0°C to about 20°C). The TFA-DCM solution may be concentrated (e.g., on a rotatory evaporator) to afford a slightly yellow oil, which may be dissolved in an alcohol (e.g., methyl alcohol (MeOH)) and water solution and then purified (e.g., via chromatography) to yield N-C5-Linker product (compound 14) as a yellow-brown oil. This N-C₅-Linker (compound 14) may be dissolved in an alcohol (e.g., methyl alcohol (MeOH)) and NMP solution to make a stock solution. [0156] Referring to FIG. 4, DCM and pyridine may be combined with 2-fluoro-5- nitroaniline (compound 15) and cooled. Phenylacetyl chloride (compound 16) may be added and solid may be formed followed by addition of water to the solution. The solid may be filtered and washed with an alcohol (e.g., MeOH) dried (e.g., under vacuum) to yield compound 17. [0157] Referring to FIG. 5, DCM and pyridine may be combined with 2-fluoro-5- nitroaniline (compound 15) and cooled. 4-methoxyphenylacetyl chloride (compound 18) may be added and a solid may be formed followed by addition of water to the solution. The solid may be filtered and washed with an alcohol (e.g., MeOH) and dried (e.g., under vacuum) to yield compound 19. [0158] Referring to FIG. 6, to produce compound (II), a mixture of N-(2-fluoro-5- nitrophenyl)benzeneacetamide (compound 17), DIPEA, and N-C5-linker (compound 14) in an alcohol (e.g., MeOH) and NMP mixed solvent may be heated at a suitable temperature (e.g., about 70°C to about 90°C) for a suitable amount of time (e.g., 2-4 days) to form a crude product. The crude product may be purified (e.g., via chromatography) to give the desired product Nitazene-N- C5-COOH (compound (II)). [0159] Referring to FIG. 7, to produce compound (III), a mixture of N-(2-fluoro-5- nitrophenyl)-4-methoxybenzeneacetamide, DIPEA, and N-C5-linker (compound 14) in an alcohol (e.g., MeOH) and NMP mixed solvent may be heated at a suitable temperature (e.g., about 70°C to about 90°C) for a suitable amount of time (e.g., 2-4 days) to form a crude product. The crude product may be purified (e.g., via chromatography) to give the desired product MeO-C5-COOH (compound (III)). [0160] Referring to FIG. 8, to produce immunogens and conjugates of Nitazene-N-C5- COOH (compound (II)), a mixture of Nitazene-N-C5-COOH (compound (II)), EDCAC, and NHS in a solvent (e.g. DMF) may be prepared to produce the activated NHS ester, Nitazene-N-C5-NHS (compound 22). [0161] Still referring to FIG.8, each protein such as, for example, OVA, BSA, KLH, and the like may be suspended in a suitable buffer and combined with Nitazene-N-C5-NHS (compound 22) in a DMF solution to produce Nitazene-N-C5-NH-OVA conjugate (compound 23a), Nitazene-N-C5-NH-BSA conjugate (compound 23b), and Nitazene-N-C5-NH-KLH immunogen (compound 23c). Reaction conditions include, for example, a buffer solution at pH of about 7 to 9, about 7.5 to 8.5, about 8. Such buffer solutions include, for example, phosphate or borate buffer etc., and combinations thereof. The resulting immunogens and conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer (PB), etc. The attachment of a protein to the linking moiety of the molecule can be through the amino group on a protein, where the nitrogen of the amino group may be the nitrogen of the linking group depicted above. [0162] Referring to FIG.9, to produce immunogens and conjugates of MeO-Nitazene-N- C5-COOH (compound (III)), a mixture of MeO-Nitazene-N-C5-COOH (compound (III)), EDCAC, and NHS in a solvent (e.g. DMF) may be prepared to produce the activated NHS ester, MeO-Nitazene-N-C5-NHS (compound 24). [0163] Still referring to FIG.9, each protein such as, for example, OVA, BSA, KLH, and the like may be suspended in a suitable buffer and combined with MeO-Nitazene-N-C5-NHS (compound 24) in a DMF solution to produce MeO-Nitazene-N-C5-NH-OVA conjugate (compound 25a), MeO-Nitazene-N-C5-NH-BSA conjugate (compound 25b), and MeO-Nitazene- N-C5-NH-KLH immunogen (compound 25c). Reaction conditions include, for example, a buffer solution at pH of about 7 to 9, about 7.5 to 8.5, about 8. Such buffer solutions include, for example, phosphate or borate buffer etc., and combinations thereof. The resulting immunogens and conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer (PB), etc. The attachment of a protein to the linking moiety of the molecule can be through the amino group on a protein, where the nitrogen of the amino group may be the nitrogen of the linking group depicted above. [0164] Referring to FIG.10, to prepare compound (IV), nitazine-4-OH (compound 6) may be dissolved in a solvent (e.g., DMF), K₂CO₃, and 3-(Boc-Amino) propyl bromide may be added. The resulting reaction mixture may be filtered off, the precipitate may be further extracted with EtOAc, and the combined organic layers may be concentrated (e.g., on a rotatory evaporator) to give a yellow oil product. The yellow oil product may be dissolved in ACN and purified (e.g., via chromatography). Fractions may be collected and concentrated (e.g., on a rotatory evaporator) to give Nitazine-O4-C3NHBoc (compound 26). [0165] Still referring to FIG. 10, to Nitazene-O4-C3NHBoc (compound 26), DCM may be added followed by TFA. The volatiles may be removed (e.g., on a rotatory evaporator) to produce Nitazene-O4-C3NH2*TFA (compound 27). Nitazene-C3NH2*TFA salt (compound 27) may be dissolved in a solvent (e.g., DMF) and NEt3 may be added followed by addition of a solution of BrAcSu in a solvent (e.g., THF). EtOAc may be added to the reaction mixture and the organic layer may be extracted with a solution of sodium phosphate monobasic, concentrated (e.g., on a rotatory evaporator), dissolved in ACN and water, purified (e.g., via liquid chromatography). Fractions may be collected and concentrated (e.g., on a rotatory evaporator) to give Nitazine-O4- C6-Br hapten (compound (IV)) [0166] Referring to FIG.11, to prepare a G6PDH conjugate with compound (IV), G6PDH enzyme may be buffer exchanged with a buffer (e.g., PB) and can be further diluted with the buffer. The enzyme solution may be blanketed with Ar and dithiothreitol (DTT) may be added followed by blanketing with argon to produce the reduced G6PDH enzyme. The reduced G6PDH enzyme may be buffer exchanged with a buffer. To the reduced G6PDH enzyme solution, Nitazene-O4-C6-Br (compound (IV)) may be added to produce Nitazene-O4-C6-3K G6PDH conjugate (compound 28). The resulting conjugate may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [0167] Alternative synetheses for producing compounds, conjugates, and immunogens encompassed by Formulas (I), (Ia), (Ib), and (Ic) are provided in FIGS.12-15. D. Antibodies [0168] Antibodies raised against the compounds described above and useful in immunoassays for the determination of nitazene are provided herein. [0169] In any embodiment, the antibodies may be raised against a complex of Formula (I) and Formula (Ia), wherein an immunogenic carrier is present (e.g., Y¹, Y², and/or Y³ may be KLH, BSA, BTG, OVA, BGG, or G6PDH). The antibodies described herein may preferentially bind to nitazene and/or a metabolite of nitazene. The preferential binding of the antibody to nitazene and/or a metabolite of nitazene may be measured by any suitable means, including but not limited to a dissociation constant (KD) of the antibody for nitazene and/or a metabolite of nitazene. [0170] Additionally or alternatively, the preferential binding of an antibody may be measured by an amount, e.g., a percentage, of nitazene recovered from a sample, such as a sample from an immunoassay. In any example, an antibody may recover at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of nitazene. [0171] The immunoassay is not particularly limited, and may be an enzyme multiplied immunoassay technique (EMIT), an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme channeling immunoassay (ECIA), a fluorescence polarization immunoassay (FPIA), an enzyme modulate mediated immunoassay (EMMIA), a substrate labeled fluorescence immunoassay (SLFIA), a combined enzyme donor immunoassay (CEDIA), a particle enhanced turbidimetric inhibition immunoassay (PETINIA), a particle enhanced turbidimetric immunoassay (PETIA), a sol particle immunoassay (SPIA), a disperse dye immunoassay (DIA), a metalloimmunoassay (MIA), an enzyme membrane immunoassays (EMIA), or a luminoimmunoassays (LIA). In a specific embodiment, the immunoassay may be an EMIT immunoassay. [0172] Methods of preparing an antibody are also provided herein. The method may include immunizing an antibody-producing animal with an immunogen comprising a composition comprising the complex of Formula (I), and isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen. E. Nucleotide Sequences [0173] The antibodies described above can be prepared by conventional means known in the art. When monoclonal antibodies are desired, the amino acid sequence can be encoded by a nucleotide sequence (e.g., a DNA sequence) and provided to a cell (e.g., a hybridoma, bacteria, yeast, etc.) which translates the nucleotide sequence to the antibody. The nucleotide sequences may be incorporated into a vector (e.g., a viral vector, a plasmid, etc.) for insertion into a cell (e.g., transformation, transfection, etc.) for subsequent production of the antibody. F. Kits [0174] Kits for determining the presence of nitazene and/or a metabolite of nitazene in a sample, such as a biosample, are also provided herein. [0175] In any embodiment, the kit may include, for example, in packaged combination, an anti-nitazene antibody described above and a conjugate of an enzyme and a nitazene analog. For example, the conjugate may correspond in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme. [0176] In any embodiment, the conjugate of (ii) may be further defined wherein when R¹ is hydrogen or CH₂CH₃ and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. For example, a compound corresponding in structure to Formula (Ib) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f) , compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0177] Additionally or alternatively, in a kit as described herein, the conjugate may correspond in structure to a Formula (Ic): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme. [0178] Examples of a suitable enzyme, when present, include, but are not limited to, glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, or horseradish peroxidase, or a functional isoform and/or analog thereof. For example, the G6PDH conjugated to the conjugate may be a putative wild-type G6PDH or a mutant or recombinant form of G6PDH so long as the mutant or recombinant G6PDH can convert glucose-6-phosphate to 6-phospho-D- glucono-1,5-lactone. The functional isoform or analog of the alkaline phosphatase or horseradish peroxidase includes mutant or recombinant alkaline phosphatases or horseradish peroxidases that catalyze the same chemical reaction as wild-type or putative alkaline phosphatase (e.g., converting p-nitrophenyl phosphate (PNP) to p-nitrophenol) or horseradish peroxidase (e.g., oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB)). [0179] In any embodiment, the conjugate of (ii) is further defined wherein when R¹ is hydrogen or CH₂CH₃ and R² is hydrogen, then R³ is not hydrogen, –OCH₃, –OCH₂CH_3, –O(CH₂)_2- CH3, –OCH(CH3)2, or –O(CH2)3CH3. For example, a compound corresponding in structure to Formula (Ic) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0180] In any embodiment, the kit may comprise reagents sufficient for a single use assay or for a multiple use assay. For example, where a single use of the assay is warranted, the kit may comprise an individually packaged volume or amount of the antibody sufficient for a single run of the assay and a volume or amount of the enzyme/nitazene analog conjugate sufficient to detect the presence of nitazene and/or a metabolite of nitazene in the sample. Where multiples uses of the assay are warranted, the kit may comprise multiple, independently packaged volumes or amounts of the antibody and volumes or amounts of the enzyme/nitazene analog conjugate. Alternatively, the components of the kit may be packaged in larger volumes of the antibody and/or the conjugates where the artisan removes only a portion of said larger volume to run the assay to determine whether the sample contains nitazene. The components of the kit may otherwise be packaged at a relatively higher concentration of the antibody and/or the conjugates where the artisan removes only a portion of said concentrated volume and dilutes said volume prior to performing the assay to determine whether the sample contains nitazene. The antibody and/or conjugate may be provided in a powdered or lyophilized form for solubilization prior to use. The kit can further include a written description of a method in accordance with the present invention as described above. G. Methods and Assays [0181] Complexes corresponding in structure to Formula (I), including where the compound comprises an enzyme, and antibodies raised against a complex corresponding in structure to Formulas (I) and (Ia) may be employed as reagents in all types of immunoassays to determine the amount (e.g., concentration) of nitazene and/or a metabolite of nitazene in samples. The reagents may also be employed in multi-analyte immunoassays wherein the presence or absence of multiple analytes may be determined. [0182] Methods for determining and/or detecting the presence of nitazene and/or a metabolite of nitazene in a sample, such as a biosample, are provided herein. In any embodiment, the method for determining and/or detecting the presence of nitazene and/or a metabolite of nitazene in a sample includes combining the sample, a conjugate of an enzyme and nitazene analog, and an anti-nitazene antibody described herein in a medium. The method further includes examining the medium for the presence of a complex comprising the anti-nitazene antibody and nitazene. In any embodiment of the method, the conjugate may correspond in structure to a Formula (Ib): an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase. [0183] In some embodiments, the conjugate may be further defined wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. For example, a compound corresponding in structure to Formula (IB) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0184] Additionally or alternatively, in a method as described herein, the conjugate may correspond in structure to a Formula (Ic): (Ic) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g are zero or h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase. [0185] In some embodiments, the conjugate may be further defined wherein when R¹ is hydrogen or –CH₂CH_3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. For example, a compound corresponding in structure to Formula (Ic) may not be one or more of compound (a), compound (b), compound (c), compound (d), compound (e), compound (f), compound (g), compound (h), compound (i), and compound (j), each as provided herein. [0186] In any embodiment, the enzyme may be as described herein. For example, the enzyme may include, but is not limited to, glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, or horseradish peroxidase. [0187] Additionally or alternatively, the method may further include: incubating the sample, conjugate, and antibody for a time sufficient for the antibody to bind to the nitazene and/or metabolite of nitazene in the sample; adding a substrate for the enzyme to the sample; and measuring the activity of the enzyme. The substrate for the enzyme may be G6PDH substrate (e.g., D-glucose 6-phosphate), an alkaline phosphatase substrate (e.g., p-nitrophenol (PNP)), or a horseradish peroxidase substrate (e.g., 3,3’,5,5’-tetramethylbenzidine (TMB)). In such a method, the presence of a complex comprising nitazene and the anti-nitazene antibody is proportional to the activity of the enzyme. [0188] The sample tested in the method is not particularly limited. It may be organic or inorganic, biological (e.g., a “biosample”), non-biological, or environmental. Examples of a biological or biosample include, but are not limited to, urine, whole blood, plasma, serum, lymph, mucus, expressed breast milk, semen, stool, sputum, cerebral spinal fluid, tears, hair, saliva, cells, tissues, an organ, and/or a biopsy. In particular, the sample may be urine, blood, plasma, mucus, or saliva. [0189] The assays described above may use various buffers to achieve and maintain a desired pH. The buffer is not particularly limited and may be borate, phosphate, carbonate, tris, barbital, and the like. Additional components, such as stabilizers for the medium, additional proteins (e.g., albumins to block non-specific and/or off-target antibody binding), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions, surfactants, and binding enhances may be used as necessary. Incubation times and temperatures (e.g., for antibody binding) are not particularly limited and may be adjusted as necessary. Incubation temperatures may be about 5°C to about 99°C, such as about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 99°C. Incubation times may be about 0.2 sec to about 6 h or overnight, for example about 5 min, about 10 min, about 15 min, about 30 min, about 45 min, about 1 h, about 1.5 h, about 2 h, about 3 h, about 4 h, about 5 h, or about 6 h. The specific time and temperature of the incubation may depend on the reagents used. [0190] The concentration of analyte that may be assayed generally varies from about 10⁻⁵ to about 10⁻⁹ M, more usually from about 10⁻⁶ to about 10⁻⁸ M. Considerations, such as whether the assay is qualitative, semi-quantitative or quantitative (relative to the amount of analyte present in the sample), the particular detection technique and the concentration of the analyte will normally determine the concentrations of the various reagents. [0191] Binding of the antibody for nitazene may result in the formation of an immune complex that can be detected directly or indirectly in numerous ways that are well known in the art. The immune complexes are detected directly, for example, when the antibodies employed are conjugated to a label. The immune complex is detected indirectly by examining for the effect of immune complex formation in an assay medium on a signal producing system. [0192] Activation of the signal producing system depends on the nature of the signal producing system members. Activation methods include for example, light activation, addition of base of pH systems, radioactivity, and addition of substrate, wherein a cofactor may be also added if necessary. [0193] In certain embodiments first and second labels may be employed and comprise a label pair. These label pairs may be, for example, a singlet oxygen generator or sensitizer and chemiluminescent reactant pair, an enzyme pair wherein a product of the first enzyme serves as a substrate for the second enzyme and a luminescent energy donor and acceptor pair, e.g., an energy donor or acceptor and a fluorescent compound. The signal will usually be initiated by and/or detected as electromagnetic radiation and will preferably be luminescence such as chemiluminescence, fluorescence, electroluminescence, or phosphorescence. [0194] The examination for presence and level of the signal also includes the detection of the signal, which is generally merely a step in which the signal is read. The signal is normally read using an instrument, the nature of which depends on the nature of the signal. The instrument may be a spectrophotometer, fluorometer, absorption spectrometer, luminometer, chemiluminometer, actinometer, photographic instrument, and the like. The presence and level of signal detected is related to the presence and amount of the entactogen/analyte present in a sample above the predetermined cut-off level. Temperatures during measurements generally range from about 10°C to about 70°C, more usually from about 20°C to about 45°C, more usually about 20°C to about 25°C. In one approach standard curves are formed using known concentrations of the analytes to be screened. Calibrators and other controls may also be used. [0195] In any embodiment, the method may be or utilize an immunoassay, such as enzyme multiplied immunoassay technique (EMIT), an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme channeling immunoassay (ECIA), a fluorescence polarization immunoassay (FPIA), an enzyme modulate mediated immunoassay (EMMIA), a substrate labeled fluorescence immunoassay (SLFIA), a combined enzyme donor immunoassay (CEDIA), a particle enhanced turbidimetric inhibition immunoassay (PETINIA), a particle enhanced turbidimetric immunoassay (PETIA), a sol particle immunoassay (SPIA), a disperse dye immunoassay (DIA), a metalloimmunoassay (MIA), an enzyme membrane immunoassays (EMIA), and a luminoimmunoassays (LIA). [0196] The EMIT assay is a homogenous enzyme immunoassay based on competition between a drug (e.g., nitazene) in the sample and the drug-conjugated to the enzyme (e.g., G6PDH). The method comprises the steps of: incubating the sample, drug-enzyme conjugate, and antibody for a time sufficient for the antibody to bind to the drug (e.g., nitazene) in the sample; adding an enzyme substrate to the sample; and measuring the activity of the enzyme, wherein the presence of the complex comprising nitazene and the antibody is proportional to the activity of the enzyme. The unbound enzyme conjugate converts the oxidized nicotinamide adenine dinucleotide (NAD⁺) to NADH and a change in absorbance at 340 nm is measured. Enzyme activity decreases upon binding to the antibody, which allows the analyte concentration in the sample to be measured in terms of enzyme activity. Enzyme activity can be measured as conventional in the art, such as measuring a change in fluorescence, radioactivity, or color of the sample. Measurement of the enzyme activity may be quantitative or qualitative. EXAMPLES General Synthesis Procedures [0197] Materials and Equipment: The compounds were purified on a Biotage LC (Charlotte, NC) equipped with a Sili cycle column (for normal phase separation) or a Shimadzu HPLC system (Riverwood, MD) equipped with a Silica-bond – C18 reverse phase column. The chemical reactions were monitored by TLC (thin layer chromatography) using Silica gel plates from Analytic Inc. (Newark, DE) and ESI-MS Waters HPLC (Milford, MA). The silica gel plates were visualized using UV short wave (254 nm). All chemicals were obtained from Sigma Aldrich (St. Louis, MO), Fluke (Waltham, MA), ThermoScientific (Waltham, MA), VWR (Radnor, PA) and used as received. ¹H NMR was recorded on a Bruker Ultra Shield™ 600 MHz spectrometer (Bruker, Billerica, MA). Chemical shifts were reported in parts per million (ppm, ^) and related to tetramethyl silane or with deuterated solvent as internal reference. NMR abbreviations used are: s (singlet), bars (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), qui (quintet) J (coupling constant), Hz (Hertz). ESI-MS spectra were recorded on a Water UPLC (Milford, MA) instrument at Siemens Healthineers RD department (Newark, DE). UV: Carry 60 was used for OD₂₈₀ and Nanodrop 2000. [0198] The following abbreviations have the meanings set forth below: 3K G6PDH – recombinant glucose-6-phosphate native enzyme ACN – acetonitrile BGG – bovine gamma globulin Boc2O – di-tert-butyl decarbonate BrAcSu - bromoacetic acid N-hydroxysuccinimide ester BSA – bovine serum albumin BTG – bovine thyroglobulin clad. – calculated CDCN-d3 – deuterated Acetonitrile having 3 deuterium atoms (for NMR spectra) CDCl₃ – deuterated Chloroform (for NMR spectra) CFA – complete Freund’s adjuvant cm² – square centimeter CV – column volume DCM – dichloromethane DI water – deionized water DIPEA - N,N-Diisopropylethylamine DMAc - Dimethylacetamide DMF – N,N-dimethylformamide DTT – Dithiothreitol EDC or EDC∙HCl – N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride eq. – molar equivalent EDTA – ethylenediaminetetraacetic acid EDTA-Na₂ – ethylenediaminetetraacetic acid disodium salt EMIT - enzyme multiplied immunoassay technique ELISA – enzyme-linked immunosorbent assay ESI-MS – electrospray ionization mass spectrometry EtOAc – ethyl acetate Fab fragment – antigen binding fragment g – grams G6P – glucose-6-phosphate G6PDH – glucose-6-phosphate dehydrogenase enzyme G₆PDNa₂ – glucose-6-phosphate disodium salt (substrate) h – hour ¹H – proton (for NMR) HAT – hypoxanthine, aminopterin, and thymidine HCl – hydrochloric acid HPLC – high-performance liquid chromatography IP - intraperitoneal KD – equilibrium dissociation constant KLH – keyhole limpet hemocyanin KU – dimeric protein complex of nG6PDH LC – liquid chromatography mAb – monoclonal antibody min – minute MeOH – methanol MeOD-d4 – deuterated methanol having 4 deuterium atoms (for NMR spectra) mg – milligram μg – microgram μL – microliter MHz – megahertz mmol, mM – millimole MWCO – molecular weight cut-off m/z – mass to charge ratio mL - milliliter NaOAc – sodium acetate NaOAc buffer – sodium acetate buffer ^-NADH – nicotinamide adenine dinucleotide NEt3 – triethylamine nm – nanometer NMR – nuclear magnetic resonance OVA – ovalbumin isolated from chicken egg white (Sigma) PB – phosphate buffer RBF – round bottom flask Rf – retention factor in TLC analysis rt – room temperature RPM – rotations per minute RU – response unit SDS-PAGE – sodium dodecyl sulphate-polyacrylamide gel electrophoresis SuOH – N-hydroxysuccinimide TBDMSCl – tert-Butyldimethylsilyl chloride THF - tetrahydrofuran TFA – trifluoroacetic acid TLC – thin-layer chromatography TRIS – tris(hydroxymethyl)aminomethane TLC – thin layer chromatography UV – ultraviolet v/v – volume ratio. Example 1: Synthesis of Compound (I) [0199] Preparation of TBDMSO-Ph-COOH (compound 2) (FIG. 1, Scheme 1): 2-(4- hydroxyphenyl)acetic acid ( compound 1) (1g, 6.58 mmol) and imidazole (1.18g) were dissolved in DCM (20 mL), then TBDMSCl (1.48 mg) was added portion wise. The resulting reaction mixture was stirred at rt for 16h. The organic layer was washed with DI water (2 x 20 mL), then evaporated to dryness. The NMR of the crude reaction mixture showed the presence of di- TBDMS-derivative. The crude product was then dissolved in THF (20 mL) and washed with Na₂CO₃ aqueous solution (1g Na₂CO₃ dissolved in 20 mL water) for 30 mins. The hydrolysis of diTBDMS-derivative was monitored by TLC (EtOAc: Hexanes 1/4 v/v, Rf of the diTBDMS compound is 0.70 and of the compound 2 is 0.4). The organic solvent was removed on a rotatory evaporator, HCl was added until pH~4, then EtOAc (30 mL) was used to extract compound 2. Compound 2 was then purified by Biotage using EtOAc/Hexanes, the product eluted with 15% EtOAc to give 1.5 g of compound 2. m/z calcd for [C14H23O3Si]+ 267.14, did not ionize on LC- MS.¹H NMR (600 MHz, CDCl₃) 7.14 – 7.12 (m, 2H), 6.80 – 6.78 (m, 2H), 3.57 (s, 2H), 0.98 (s, (s, 2H), 0.19 (s, 6H), 0.10 (s, 1.51H). [0200] Preparation of TBDMSO-Ph-COCl (compound 3): To an oven dried flask equipped with a magnetic stir bar, TBDMSO-Ph-COOH (compound 2) (424 mg, 1.59 mmol) was dissolved in DCM (10mL), DMF (2 drops, cat.) was added followed by oxalyl chloride (500 µL). The resulting reaction mixture was blanketed with argon and stirred at rt. for 90 mins. The volatiles were removed on a rotatory evaporator and the resulting oil was further dried on an oil pump for 40 mins to produce compound 3. THF (5 mL) was then added to form TBDMS-Ph-COCl-THF solution. [0201] Preparation of compound 4: To an oven dried flask equipped with a stir bar, 2- fluoro-5-nitroaniline (248 mg, 1.59 mmol) was dissolved in THF (10 mL), then NEt3 (2eq. 442 µL) was added. The resulting reaction mixture was stirred for 5 mins, then TBDMS-Ph-COCl- THF solution prepared previously was added drop wise over 2 mins. The resulting reaction mixture was blanketed with argon, and a yellow precipitate was formed. Additional THF (20 mL) was used, however, it did not improve the solubility. The yellow solid was filtered off and the filtrate was mixed with Silica gel (0.5 g) and dried on a rotatory evaporator and purified by normal phase (Biotage) using EtOAc/Hexanes. Fractions containing the product were collected and concentrated to give compound 4186 mg (0.46 mmol, in 29 % yield) as a yellow powder. M/z calcd for [C20H26FN2O4Si]+ ACN 446.19, found 446.50 (major peak as ACN adduct), 405.50 minor fragmentation, ret. Time 6.010 mins. [0202] Preparation of Nitazene-4-OH (compound 6): To a glass vial, compound 4 (180 mg, 445 mmol) was dissolved in DMAc (2mL), then, N,N-Diethyl-ethylenediamine (100 µL, 2 eq.) and DIPEA (130 µL) were added. The resulting reaction mixture was blanketed with argon and stirred at 40°C for 16h. The uPLC showed that the fluoride anion generated during the SNAr reaction deprotected the TBDMS group, therefore, compound 5 was not isolated, the reaction was then heated to 70-100°C for 3 days yielding compound 6 exclusively. DMAc was evaporated on a rotatory evaporator, then EtOAc (20 mL) was added and washed with brine (20 mL), followed by DI water (20 mL). The solvent was removed in vacuum to give 186 mg of crude Nitazene-4- OH (compound 6) (quant. yield) as yellow precipitate. m/c calcd for [C20H25N4O3]+ 369.19, found 369.57, ret. time 2.014. [0203] Preparation of Nitazene-O4-C5-OtBu (compound 7): Nitazene-4-OH (compound 6) (59 mg, 0.126 mmol) was dissolved in DMF (700 µL) and K₂CO₃ (70 mg, 0.506 mmol) and tert-butyl-Bromo valerate (2.5 eq.) were added. The resulting reaction mixture was stirred at 60°C overnight. The resulting reaction mixture was filtered off, the precipitate was further extracted with EtOAc (1 mL), and the combined organic layers were concentrated on a rotatory evaporator to give a yellow oil which was dissolved in ACN to a total volume of 2 mL and purified by Shimadzu HPLC. [0204] Solvent A: Water +0.1% AcOH, Solvent B: Acetonitrile (ACN) + 0.1% AcOH. [0205] Shimadzu method: 0 – 2 min isocratic Solvent B 10%, 2 – 20 min gradient Solvent B 70%, 20 – 24 mins isocratic Solvent B 70%, 24 – 27 min gradient Solvent B 10%, 27 – 28 mins isocratic Solvent B 10%. [0206] Fractions containing the useful product were pooled, concentrated, and lyophilized to give 30.5 mg (eluting at Solvent B 50%) of compound 7, while fractions 9,11 contained the starting material (18 mg, eluting using Solvent B 33%). m/z calcd. for [C29H41N4O5]+ 525.31, found 525.58., ret. Time 3.208 min. [0207] 1H NMR (600 MHz, CDCl3): 8.66 (d, J = 2.02 Hz, 1H), 8.20 (q, J = 2.12 Hz, 1H), 7.38 (d, J = 8.88 Hz, 1H), 7.15 (d, J = 8.59 Hz, 2H), 6.84 (d, J = 8.60 Hz, 2H), 4.34 (s, 2H), 4.14 (t, J = 6.91Hz, 2H), 3.93 (t, J = 5.83 Hz, 2H), 2.55 (t, J = 6.95 Hz, 2H), 2.50 (q, J = 7.13 Hz, 4H), 2.28 (t, J = 7.33 Hz, 2H), 1.80 – 1.75 (m, 4H), 1.44 (s, 9H), 0.90 (t, J = 7.12 Hz, 6H). [0208] Preparation of compound (I): Compound 7 (30.5 mg, 0.058 mmol) was suspended in DCM/TFA v/v 100/140 µL and stirred at rt. for 2h. The volatiles were removed in vacuo to give Nitazene-O4-C5-OH (compound (I)) (27 mg, 0.058 mmol), in quantitative yield. DMF (540 µL) was added to make a 50 mg/mL stock solution. Example 2: Preparation of Immunogens and Conjugates of Compound (I) [0209] Preparation of Compound (9) (FIG. 2, Scheme 2): Nitazene-O4-C5-OH (compound (I)) (17 mg, 0.036 mmol) as 340 µL DMF stock solution was placed onto an oven dried vial. EDC*HCl (11.22 mg, 0.058 mmol) and SuOH (8 mg, 0.069 mmol) were then added. The resulting reaction mixture was stirred overnight, the next day it was warmed up to 40°C for 5h to complete the reaction to yield activated Nitazene-O4-C5-OSu (compound (9). The formation of the activated Nitazene-O4-C5-OSu was confirmed by uPLC MS, m/z for [C29H36N5O7]+ 566.26, found 566.56. DMF (340 µL) was added to make a 25 mg/mL activated hapten-DMF solution. [0210] Buffer used: Buffer 1 (50 mM PB pH 8.00) and Buffer 2 (50 mM PB pH 7.00). These buffers are prepared on a 2 L scale. Buffer 3 (50 mM PB pH 7.25 + 2.5 mM EDTA), Buffer 4 [Buffer 3 (400 mL + 20 µL of DTT (0.5 M))]. [0211] Preparation of Nitazene-O4-OVA, BSA, KLH conjugates and immunogens (compounds 10a, 10b, 10c) (FIG. 2, Scheme 2a): OVA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 196 µL of the Nitazene-O4-C5-OSu- DMF solution (4.89 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the hapten was added. DMF (0.8 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 16h. The next day, it was purified by G25M Sephadex column (CV = 60 mL) using Buffer 2. A fraction of 15 mL containing the NitazeneO4-C5-OVA conjugate (compound 10a) was collected, which was then concentrated to 8.50 ml (c = 1.55 mg/mL). The conjugate was filtered through CA filters (0.20 um). [0212] BSA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 202 µL of the Nitazene-O4-C5-OSu-DMF solution (5.05 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the hapten was added. DMF (0.8 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 16h. The next day, it was purified by G25M Sephadex column (CV = 60 mL) using Buffer 2. A fraction of 15 mL containing the NitazeneO4-C5-BSA conjugate (compound 10b) was collected, which was then concentrated to 3.70 ml (c = 3.40 mg/mL). The conjugate was filtered through CA filters (0.20 um). [0213] KLH (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 0.292 µL of the Nitazene-O4-C5-OSu-DMF solution (7.32 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the hapten was added. DMF (0.8 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 16h. The next day, it was purified by G25M Sephadex column (CV = 60 mL) using Buffer 2. A fraction of 15 mL containing the NitazeneO4-C5-KLH immunogen (compound 10c) was collected, which was then concentrated to 4.50 ml (c = 3.42 mg/mL). The immunogen was filtered through CA filters (0.20 um). [0214] Preparation of Nitazene-O4-G6PDH conjugates (compounds 10d, 10e, 10f, 10g, and 10h) (FIG. 2, Scheme 2b): Nitazene-O4-C5-OH (compound (I)) (5 mg, 0.010 mmol) as 100 µL DMF stock solution was placed onto an oven dried vial. EDC*HCl (3 mg, 0.016mmol) and SuOH (2.3 mg, 0.02 mmol) were then added. The resulting reaction mixture was stirred overnight. The formation of the activated Nitazene-O4-C5-OSu (compound 9) was confirmed by uPLC MS, m/z for [C29H36N5O7]+ 566.26, found 566.56. DMF (900 µL) was added to make a 5 mg/mL activated hapten-DMF solution. [0215] G6PDH (30 mg) was dialyzed with Buffer 1 (2 x 250 mL) in a cold room. After dialysis, the concentration was adjusted to 5 mg/mL using Buffer 1 then G6PDNa (30 mg) and β- NADH were added. The mixture was rocked until dissolved then the enzyme was placed in 5 vials (5mg, 1 mL each). The vials were chilled on an ice bath, then the activated hapten-DMF solution corresponding to 5x to 25x was added dropwise (column 2 of Table 2). DMF (100 µL) was also added to enhance the solubility. The resulting reaction mixtures were then stirred at rt overnight. The next day, they were purified by G25M Sephadex column (CV~ 30 mL) using Buffer 2. A fraction of 15 mL containing the G6PDH conjugate was collected form each run and concentrated on Amicon stir cells (MW CO 10,000) to the volumes indicated in Column 3 of Table 2 below to form compounds 10d, 10e, 10f, 10g, and 10h; each conjugate’s concentration is estimated by NanoDrop2000 measurement (Column 4 in Table 2) Table 2 Conjugate ID mhapt [mg] | vhapten [µL] V [mL] c [mg/mL] Example 3: Synthesis of Compounds (II) and (III) [0216] Preparation of N-C₅-Linker (compound 14) (FIG. 3, Scheme 3): A mixture of K2CO3 (1221 mg), 1-(Boc-amino)-2-(ethylamino)ethane solid (compound 11) (750 mg), and tert- Butyl 5-bromovalerate (compound 12) (1.88 g, 2.05 mL) in acetonitrile (12 mL) was heated at 80°C for 20 hours while stirring. The reaction was cooled to rt. The solid was filtered and washed with ACN (3×5 mL). The combined acetonitrile filtrate was concentrated on a rotatory evaporator to afford a colorless solid as compound 13. The solid (compound 13) was treated with 1:1 TFA- DCM (20 ml) (0 °C to rt) over night. The DCM-TFA solution was concentrated on a rotatory evaporator to afford a slightly yellow oil. This oil crude product was dissolved in 9:1 MeOH-water (10 mL), then purified by strong cation exchange (SCX). The pure N-C₅-Linker product (compound 14) was obtained as a yellow-brown oil (750 mg). M/z calcd for [C9H21N2O2]⁺ 189.16, found 189.20. This N-C₅-Linker was dissolved in 1:6 MeOH-NMP (15 mL) to make a stock solution (50 mg/mL, 750 mg/15 mL). [0217] Preparation of N-(2-fluoro-5-nitrophenyl)benzeneacetamide (compound 17) (FIG. 4, Scheme 4): To a 100 mL size two-neck RBF charged with 2-fluoro-5-nitroaniline (compound 15) (652 mg) and a stir bar, DCM (15 mL) and pyridine (0.70 mL) were added. The RBF was cooled on an ice-bath for 30 minutes. Phenylacetyl chloride (compound 16) (770 mg) was added to the RBF through a syringe. Precipitate came out from the solution. The reaction was stirred overnight from ice-water cold to rt. Next day, water (0.50 mL) was added. The resulting mixture was stirred for 3 hours. The solid was filtered and washed with MeOH (3x5 mL). The solid was checked by LCMS and indicated it was the desired product, and it was pure. M/z calcd for [C14H12FN2O3]+ ACN 316.11, found 316.21 (major peak as ACN adduct). The solid was dried under vacuum overnight to afford the desired product (compound 17) as a white-gray solid (983 mg). [0218] Preparation of N-(2-fluoro-5-nitrophenyl)-4-methoxybenzeneacetamide (19) (FIG. 5, Scheme 5): To a 100 mL size two-neck RBF charged with 2-fluoro-5-nitroaniline (compound 15) (915 mg) and a stir bar, DCM (20 mL) and pyridine (1.0 mL) were added. The RBF was cooled on an ice-bath for 30 minutes. 4-methoxyphenylacetyl chloride (compound 18) (1310 mg) was added to the RBF through a syringe. Precipitate came out from the solution. The reaction was stirred overnight from ice-water cold to rt. Next day, water (0.50 mL) was added. The resulting mixture was stirred 3 hours. The solid was filtered and washed with MeOH (3x5 mL). The solid was checked by LCMS and indicated it was the desired product and it was pure. M/z calcd for [C15H14FN2O4]+ ACN 346.12, found 346.26. The solid was dried under vacuum overnight to afford the desired product (compound 19) as a white-gray solid (1185 mg). [0219] Preparation of Nitazene-N-C5-COOH (compound (II)) (FIG. 6, Scheme 6): A mixture of N-(2-fluoro-5-nitrophenyl)benzeneacetamide (compound 17) (110 mg), DIPEA (0.1 mL), and N-C5-linker (compound 14) (4.0 mL @25 mg/mL in 1:6 MeOH-NMP mixed solvent) was heated at 70°C for 2 days and then heated at 90°C for 2 days. After heating 4 days, LCMS indicated no more of compound 17 remained and the desired product was observed as the major product. After cooling to rt, the crude product solution was directly purified by HPLC. The desired fractions were collected and lyophilized to give the desired product Nitazene-N-C5-COOH (compound (II)) as a white solid (139 mg). M/z calcd for [C23H29N4O4]+ 425.34, found 425.2. [0220] Preparation of MeO-Nitazene-N-C5-COOH (compound (III)) (FIG.7, Scheme 7): A mixture of N-(2-fluoro-5-nitrophenyl)-4-methoxybenzeneacetamide (compound 19) (65 mg), DIPEA (0.1 mL), and N-C5-linker (compound 14) (2.2 mL at 25 mg/mL in 1:6 MeOH-NMP mixed solvent) was heated at 70°C for 2 days and then heated at 90°C for 2 days. After heating 4 days, LCMS indicated no more compound 19 remained and the desired product was observed as the major product. After cooling to rt, the crude product solution was directly purified by HPLC. The desired fractions were collected and lyophilized to give the desired product MeO-C5-COOH (compound (III)) as a white solid (78 mg). M/z calcd for [C24H31N4O5]+ 455.23, found 455.47. Example 4: Preparation of Immunogens and Conjugates of Compounds (II) and (III) [0221] Preparation of Nitazene-N-C5-NHS (compound 22) (FIG.8, Scheme 8): A mixture of Nitazene-N-C5-COOH (compound (II)) (28.8 mg) EDCAC (26.1 mg), and NHS (15.6 mg) in DMF (1.2 mL) was stirred for 6h. After this period, LCMS indicated >90% carboxylic acid was converted to its NHS ester (compound 22). This DMF solution (24 mg/mL hapten) was directly used in the next step bio-conjugation. [0222] Preparation of Nitazene-N-C5-OVA, BSA, KLH immunogens and conjugates (compounds 23a, 23b, and 23c) (FIG.8, Scheme 8): OVA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 310 µL of the Nitazene-N-C5-NHS (compound 22) in DMF solution (7.3 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL mm) using Buffer 2. A fraction of 12 mL containing the Nitazene-N-C5-NH-OVA conjugate (compound 23a) was collected which was then concentrated from 12 mL to 5.3 mL. The conjugate was filtered through a CA filter (0.20 um) to give the final conjugate (5.1 mL). [0223] BSA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 315 µL of the Nitazene-C5-NHS (compound 22) in DMF solution (7.6 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became slightly cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL) using Buffer 2. A fraction of 12 mL containing the Nitazene-N-C5-NH-BSA conjugate (compound 23b) was collected, which was then concentrated to 5.1 mL. The conjugate was filtered through a CA filter (0.20 um) to give the final conjugate (4.9 mL). [0224] KLH (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 343 µL of the Nitazene-C5-NHS in DMF solution (8.2 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL) using Buffer 2. A fraction of 12 mL containing the Nitazene-N- C5-NH-KLH immunogen (compound 23c) was collected, which was then concentrated to 5.3 mL. The conjugate was filtered through a CA filter (0.20 um) to give the final conjugate (5.1 mL). [0225] Buffer used: Buffer 1 (50 mM PB pH 8.00) and Buffer 2 (50 mM PB pH 7.00). These buffers were prepared on a 2 L scale. [0226] Preparation of MeO-Nitazene-N-C5-NHS (compound 24) (FIG. 9, Scheme 9): A mixture of MeO-Nitazene-N-C5-COOH (compound (III)) (30.8 mg) EDCAC (26.1 mg), and NHS (18.5 mg) in DMF (1.2 mL) was stirred for 40h. After this period, LCMS indicated all carboxylic acid was converted to its NHS ester, MeO-Nitazene-N-C5-NHS (compound 24). This DMF solution (25.7 mg/mL hapten) was directly used in the next step bio-conjugation. [0227] Buffer used: Buffer 1 (50 mM PB pH 8.00) and Buffer 2 (50 mM PB pH 7.00). These buffers are prepared on a 2 L scale. [0228] Preparation of MeO-Nitazene-N-C5-OVA, BSA, KLH immunogens and conjugates (compounds 25a, 25b, and 25c) (FIG. 9, Scheme 9): OVA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 310 µL of the MeO- Nitazene-N-C5-NHS (compound 24) in DMF solution (8.0 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL mm) using Buffer 2. A fraction of 12 mL containing the MeO-Nitazene-N- C5-NH-OVA conjugate (compound 25a) was collected which was then concentrated from 12 mL to 5.1 mL. The conjugate was filtered through a CA filter (0.20 µm) to give the final conjugate (4.9 mL). [0229] BSA (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 315 µL of the MeO-Nitazene-C5-NHS (compound 24) in DMF solution (8.1 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became slightly cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL) using Buffer 2. A fraction of 12 mL containing the MeO-Nitazene-N-C5-NH-BSA conjugate (compound 25b) was collected, which was then concentrated to 5.3 mL. The conjugate was filtered through a CA filter (0.20 um) to give the final conjugate (5.1 mL). [0230] KLH (20 mg) was suspended in Buffer 1 (4 mL). The resulting mixture was chilled on an iced bath, then 343 µL of the MeO-Nitazene-C5-NHS in DMF solution (8.2 mg of activated hapten) was added dropwise over 5 mins. The reaction mixture became cloudy as the activated hapten was added. DMF (0.5 mL) was also added to improve the solubility. The resulting reaction mixture was allowed to warm up to rt and stirred for 18h. The next day, it was purified by G25M Sephadex column (CV = 55 mL) using Buffer 2. A fraction of 12 mL containing the MeO- Nitazene-N-C5-NH-KLH immunogen (compound 25c) was collected, which was then concentrated to 5.3 mL. The immunogen was filtered through a CA filter (0.20 um) to give the final immunogen (5.1 mL). Example 5: Synthesis of Compound (IV) [0231] Preparation of Nitazene-O4-C3NHBoc (compound 26) (FIG. 10, Scheme 10): Nitazine-4-OH (compound 6) (60 mg, 0.16 mmol) was dissolved in DMF (700 µL) and K₂CO₃ (88 mg, 0.0.64 mmol) and 3-(Boc-Amino) propyl bromide (55 mg, 0.23 mmol, 1.43 eq.) were added. The resulting reaction mixture was stirred at 60°C overnight. The resulting reaction mixture was filtered off, the precipitate was further extracted with EtOAc (1 mL), and the combined organic layers were concentrated on a rotatory evaporator to give a yellow oil, which was dissolved in ACN to a total volume of 2 mL and purified on a Shimadzu LC equipped with a C18 column. [0232] Solvent A: Water +0.1% AcOH, Solvent B: Acetonitrile (ACN) + 0.1% AcOH; UV detection 309 nm. [0233] LC method: 0 – 2 min isocratic Solvent B 10%, 2 – 20 min gradient Solvent B 70%, 20 – 24 mins isocratic Solvent B 70%, 24 – 27 min gradient Solvent B 10%, 27 – 28 mins isocratic Solvent B 10%. [0234] Fractions eluting at 14 -16.5 mins, were collected, concentrated on a rotatory evaporator to give 20 mg of Nitazine-O4-C3NHBoc (compound 26) (0.038 mmol, 24% yield) as a colorless powder. m/z calcd for [C28H40N5O5+] 526.30, found 526.49. [0235] 1H NMR (600 MHz, CDCl₃): 8.63 (d, J = 2.1 Hz, 1H), 8.17 (dd, J = 8.86, 2.1 Hz, 1H), 7.34 (d, J = 8.86 Hz, 1H), 7.14 ( d, J = 8.66 Hz, 2H), 6.83 (d, J = 8.66 Hz, 2H), 4.75 (s, 1H), 4.33 (s, 2H), 4.09 (t, J = 6.7 Hz, 2H), 3.97 (t, J = 6.00 Hz, 2H), 3.30-3.29 (m, 2H), 2.55 (t, J = 6.69 Hz, 2H), 2.47 (q, J = 7.13 Hz, 4H), 1.95 (J = 6.16 Hz, 2H), 1.41 (s, 9H), 0.87 (t, J =7.12 Hz, 6H). [0236] Preparation of Nitazene-O4-C3NH2*TFA (compound 27): To Nitazene-O4- C3NHBoc (compound 26) (20 mg, 0.038 mmol), DCM (150 µL) was added followed by TFA (120 µL). The resulting reaction mixture was stirred at rt for 4h. The LC MS confirmed the formation of Nitazene-O4-C3NH2 intermediate; m/z calcd for [C23H32N5O3+] 426.25, found 426.37. The volatiles were removed on a rotatory evaporator to give 21 mg of Nitazene-O4- C3NH2*TFA (compound 27) in quantitative yield. [0237] Preparation of Nitazene-O4-C6-Br hapten (compound (IV)): In a vial equipped with a stir bar, Nitazene-C3NH2*TFA salt (compound 27) (21 mg, 0.038 mmol) was dissolved in DMF (200 µL), NEt3 (16 µl, 3 eq.) was added, and the resulting mixture was chilled on an ice bath. Then a solution of BrAcSu (86.8 µL, 8.68 mg, 0.036 mmol, as 10 mg/mL in THF) was added dropwise. The resulting reaction mixture was stirred for 30 mins, and it was completed by LC MS. m/z calcd for [C25H33BrN5O4+] 546.17, 548.17 (as major Br isotope isomers), found 546.30, 548.28. [0238] EtOAc (10 mL) was added to the reaction mixture, the organic layer was then extracted with a solution of sodium phosphate monobasic (2 x 10 mL). The organic layer was concentrated on a rotatory evaporator and then it was dissolved in ACN/H2O 9/1 v/v (2 mL) and purified on a preparative Shimadzu LC equipped with reverse phase (C18 column). [0239] Solvent A: Water +0.05% TFA, Solvent B: Acetonitrile (ACN) + 0.05% TFA; UV detection 309 nm. [0240] LC method: 0 – 2 min isocratic Solvent B 10%, 2 – 20 min gradient Solvent B 70%, 20 – 24 mins isocratic Solvent B 70%, 24 – 27 min gradient Solvent B 10%, 27 – 28 mins isocratic Solvent B 10%. [0241] Fractions eluting at 12-15 mins were collected and concentrated on a rotatory evaporator to give 23.6 mg of Nitazine-O4-C6-Br hapten (compound (IV)) (0.038 mmol, 24% yield) as a colorless powder. m/z calcd for [C28H40N5O5+] 526.30, found 526.49. [0242] 1H NMR (600 MHz, CD3CN) 8.59 (d, J = 1.58 Hz, 1H), 8.26 (q, J = 1.93 Hz, 1H), 7.84 (d, J = 8.96 Hz, 1H), 7.25 (d, J = 8.55 Hz, 2H), 6.91 (d, J = 8.63 Hz, 2H), 4.72 (t, J = 8.26 Hz, 2H), 4.39 (s, 2H), 4.00 (t, J = 6.08 Hz, 2H), 3.79 (s, 2H), 3.34 (q, J = 6.57 Hz, 2H), 3.18 - 3.12 (m, 4H), 3.08-3.07 (m, 2H), 1.92-1.90 (m, 3H), 1.22 (t, J = 7.25 Hz, 6H). Example 6: Preparation of Conjugates of Compound (IV) [0243] The G6PDH enzyme preparation (FIG. 11, Scheme 11): The G6PDH enzyme (5 mg at 5 mg/mL) was loaded onto a dialysis bag and buffer exchanged with Buffer 3 (50 mM PB pH 7.252.5 mM EDTA) 2 x 250 mL in a cold room. The enzyme was then recovered (1 mL), and the concentration was checked at 40x dilution. [0244] The G6PDH enzyme DTT reduction: The G6PDH enzyme (1 mL) was placed into a 2 mL plastic tube, chilled on an ice bath, and blanketed with Ar. Then 12 µL of DTT (0.5 M solution) was added and the resulting mixture was blanketed again with Ar. The enzyme solution was then rocked in a cold room for 16h, to produce the reduced G6PDH enzyme. [0245] Buffer 4 Preparation: To Buffer 3 (400 mL), DTT (20 µL, 0.5M) was added. The resulting mixture was blanketed with argon then stored in a cold room overnight. The next day Buffer 2 was degassed using a vacuum line. [0246] A Sephadex G25 M column (25 ml) was equilibrated with Buffer 2 for 3 CVs. Then the reduced G6PDH enzyme was buffer exchanged on it. A volume of 7 mL of reduced enzyme was collected which was then concentrated to 1.2 mL on an Amicon Stir cell (MW CO 30 kDa cut-off 5mL) on a centrifuge at 3200 RPM at 4°C. The reduced enzyme was then placed in a glass vial equipped with a stir bar, blanketed with Ar and placed on an ice bath. [0247] Preparation of Nitazene-O4-C6-3K G6PDH conjugate (28) (FIG.11, Scheme 11): To the reduced G6PDH enzyme solution produced previously, Nitazene-O4-C6-Br (compound (IV)) (240 µL, 1.20 mg, 5 mg/mL in DMF, 40 mol excess) was added dropwise. The reaction mixture became cloudy as the hapten was added. The resulting reaction mixture was blanketed with Ar and stirred in a cold room overnight. The conjugate was purified on a Sephadex G25M column (60 ml) using Buffer 2 (50 mM PB pH 7.00) and a fraction of 15 mL of Nitazene-O4-C6- 3K G6PDH conjugate (compound 28) was collected, which was concentrated on an Amicon Stir cell (MW CO 30 kDa cut-off 15 mL) to 3.2 mL (conc.1.06 mg/mL). Example 7: EMIT Nitazene Assay [0248] An anti-nitazene antibody as described herein and a G6PDH conjugate as described herein are evaluated in an EMIT assay format. The EMIT format assay is a homogenous enzyme immunoassay technique used for the analysis of specific compounds in a biological sample. The assay is based on competition for antibody binding sites between a drug in a sample and the drug labelled with a marker, such as G6PDH. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity. When the marker is G6PDH, the enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate (G6P), resulting in an absorbance change that is measured spectrophotometrically. Endogenous G6PDH does not interfere with the assay because the coenzyme NAD functions only with the bacterial enzyme (from Leuconostoc mesenteroides) employed in the assay. The assay reactions are shown below: [0249] The EMIT reagents include one G6PDH conjugate and one anti-nitazene antibody (Ab) reagent. The G6PDH conjugate is labeled with a hapten as described herein through a linkage. In the absence of nitazene and its metabolites (“analyte”), the G6PDH conjugate is bound by the antibody (see above). The bound G6PDH in the reaction vessel generates enzymatic inhibition and decreases the signal (NADH absorption in 340 nm). The addition of nitazene and/or its metabolites from a sample to the reaction competes with the binding of the G6PDH conjugate to the antibody, resulting in disrupting enzymatic inhibition with an increase in signal generation. This increase in (NADH) signal is a direct function of the amount of nitazene and/or its metabolites in the sample when measured against a calibration curve. [0250] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure. NON-LIMITING ILLUSTRATIVE EMBODIMENTS [0251] Illustrative Embodiment 1. A complex corresponding in structure to a Formula (I): (I) wherein: ; hydrogen, an alkyl, an alkoxy, an ; X¹ is a halo; a, c, e, g, i, and k are each zero or 1; b, d, f, h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. [0252] Illustrative embodiment 2. The complex of illustrative embodiment 1, wherein the alkyl is a C₁-C₄ alkyl, preferably a C₁-C₂ alkyl; the alkoxy is a C₁-C₄ alkoxy, preferably a C₁-C₂ alkoxy; each halo is Cl or Br, preferably Br; b, d, f, h, j, and l are each 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide, wherein the protein is preferably selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6- phosphate dehydrogenase (G6PDH). [0253] Illustrative Embodiment 3. The complex of illustrative embodiment 1 or 2, wherein the complex corresponds in structure to one of Formulas (A)-(S): , , N Y_{1 ,} , ,

, , , , or wherein: R² is hydrogen, R³ is hydrogen, –OCH3, –OCH2CH3, –O(CH2)2CH3, or – OCH(CH₃)₂; m is zero to 100, preferably 1 to 25, more preferably 1 to 10. n is zero to 100, preferably 1 to 25, more preferably 1 to 10; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label, preferably wherein the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide, preferably wherein the protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH). [0254] Illustrative Embodiment 4. The complex of illustrative embodiment 1, wherein the complex is a compound corresponding in structure to a Formula (Ia):

(Ia) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , –O–, or –NH–; X¹ is a halo; a and e are each zero or 1; b and f are each zero to 100; and wherein when R¹ is hydrogen or –CH₂CH_3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3. [0255] Illustrative Embodiment 5. The complex of illustrative embodiment 4, wherein R¹ a C₁-C₄ alkyl, preferably a C₁-C₂ alkyl; R² is hydrogen; R³ is hydrogen, C_{1-C4 ; R4 is –CH2–, –O–, or –NH–; R6} is –CH2–, is 1 to 25, preferably 1 to 10, more preferably 1 to 5; e is zero or 1, preferably 1; f is 1 to 25, preferably 1 to 10, more preferably 1 to 5; and X¹ is Cl or Br, preferably Br. [0256] Illustrative Embodiment 6. The complex of illustrative embodiments 4 or 5, wherein the complex is one of the following compounds: the co pe s a cojugae co espo g s ucue o a o ua : wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. [0258] Illustrative Embodiment 8. The complex of illustrative embodiment 1, wherein the complex is a conjugate corresponding in structure to a Formula (Ic): wherein: R¹, R², and R³ are an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃. [0259] Illustrative Embodiment 9. The complex of illustrative embodiment 8, wherein R¹ is or C1-C4 alkyl, preferably C1-C2 alkyl; R² is hydrogen; R³ is , hydrogen, or a C₁-C₄ alkoxy; R⁷ is preferably–O–; g is zero or 1; h is 1 to 25, preferably 1 to 10, more preferably 1 to 5; k is zero or 1, preferably 1; and l is 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide. [0260] Illustrative Embodiment 10. The complex of illustrative embodiments 8 or 9, wherein the conjugate corresponds in structure to: , limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH). [0261] Illustrative Embodiment 11. A kit comprising: (i) an anti-nitazene antibody, preferably wherein the anti-nitazene antibody is raised against the complex of illustrative embodiments 1-3 and 7-10 wherein the immunogenic carrier is present; and (ii) a conjugate corresponding in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6- phosphate dehydrogenase (G6PDH); and optionally, wherein when R¹ is hydrogen or CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)_3- CH3. [0262] Illustrative Embodiment 12. The kit of illustrative embodiment 11, wherein the conjugate corresponds in structure a Formula (Ic): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH); and optionally, wherein when R¹ is hydrogen or CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)_3- CH3. [0263] Illustrative Embodiment 13. A method of detecting the presence of nitazene and/or a metabolite of nitazene in a sample, the method comprising the steps of: combining the sample, a conjugate, and an anti-nitazene antibody in a medium preferably wherein the anti-nitazene antibody is raised against the complex of illustrative embodiments 1-3 and 7-10 wherein the immunogenic carrier is present; and examining the medium for the presence of a complex comprising nitazene and the anti- nitazene antibody, wherein the conjugate corresponds in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6- phosphate dehydrogenase (G6PDH); and optionally, wherein when R¹ is hydrogen or –CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or – O(CH2)3CH3. [0264] Illustrative Embodiment 14. The method of illustrative embodiment 13, wherein the conjugate corresponds in structure a Formula (Ic): an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH); and optionally, wherein when R¹ is hydrogen or –CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or – O(CH2)3CH3. [0265] Illustrative Embodiment 15. A method of preparing an antibody, the method comprising: immunizing an antibody-producing animal with an immunogen comprising a composition comprising the complex of any one of illustrative embodiments 1-10; and isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen.

Claims

CLAIMS What is claimed is: 1. A complex corresponding in structure to a Formula (I): wherein: ; hydrogen, an alkyl, an alkoxy, an ; X¹ is a halo; a, c, e, g, i, and k are each zero or 1; b, d, f, h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH₂CH_3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3CH3.

2. The complex of claim 1, wherein the alkyl is a C1-C4 alkyl, preferably a C1-C2 alkyl; the alkoxy is a C₁-C₄ alkoxy, preferably a C₁-C₂ alkoxy; each halo is Cl or Br, preferably Br; b, d, f, h, j, and l are each 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide.

3. The complex of claim 2, wherein the protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH).

4. The complex of claim 1, wherein the complex corresponds in structure to one of Formulas (A)-(S): , , , , N N H N N B N m r O O₂N N NO₂ N O R³ _{O NH Formula (I)} n _{Y1 , Formula (J) ,} , , O NH n N Y¹ N O₂N N R³ F_{ormula (M) , ,} , , , , or R² is hydrogen; R³ is hydrogen, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, or –OCH(CH₃)₂; m is zero to 100, preferably 1 to 25, more preferably 1 to 10. n is zero to 100, preferably 1 to 25, more preferably 1 to 10; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label.

5. The complex of claim 4, wherein the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide, preferably wherein the protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH).

6. The complex of claim 1, wherein the complex is a compound corresponding in structure to a Formula (Ia): wherein: 1 ^{2 3} R , R , and R are an alkyl, an alkoxy, , –O–, or –NH–; a a and e are each zero or 1; b and f are each zero to 100; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃.

7. The complex of claim 6, wherein the alkyl is a C₁-C₄ alkyl, preferably a C₁-C₂ alkyl; the alkoxy is a C1-C4 alkoxy, preferably a C1-C2 alkoxy; each halo is Cl or Br, preferably Br; and b and f are each 1 to 25, preferably 1 to 10, more preferably 1 to 5.

8. The complex of claim 6, wherein R¹ is a C1-C4 alkyl, preferably a C1-C2 alkyl; R² is hydrogen; R³ ; R⁴ is –CH2–, –O–, or –NH–, preferably –O–; a is zero or 1, preferably 1 to 10, more preferably 1 to 5.

9. The complex of claim 6, wherein R¹ is ; R⁴ is –CH₂–, –O–, or – NH–; a is zero or 1, preferably zero; b is 1 10, more preferably 1 to 5; R² is 3 hydrogen; and R is hydrogen or C₁-C₄

10. The complex of claim 6, wherein R¹ is a C1-C4 alkyl, preferably a C1-C2 alkyl; R² is hydrogen; R³ ; R⁶ is –CH2–, –O–, or –NH–, preferably –O–; e is zero or 1, 1 to 10, more preferably 1 to 5; and X¹ is Cl or Br, preferably Br.

11. The complex of claim 6, wherein the complex is one of the following compounds:

12. The complex of claim 1, wherein the complex is a conjugate corresponding in structure to a Formula (Ib): (Ib) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, are zero or h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an immunogenic carrier or a label; and wherein when R¹ is hydrogen or –CH2CH3, and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃.

13. The complex of claim 1, wherein the complex is a conjugate corresponding in structure to a Formula (Ic): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , R⁷ and R⁹ are each independently –CH2–, –O–, or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an immunogenic carrier or a label; and wherein when R¹ is –CH3 or –CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)₃CH₃.

14. The complex of claim 13, wherein R¹ is C₁-C₄ alkyl, preferably C₁-C₂ alkyl, R² is hydrogen; R³ ; R⁷ is –CH₂–, –O–, or –NH–, preferably –O–; g is zero or 1, 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide.

15. The complex of claim 13, wherein R¹ ; R⁷ is –CH2–, –O–, or –NH– ; g is zero or 1, preferably zero; h is 1 ² more preferably 1 to 5; R is hydrogen; R³ is hydrogen or a C₁-C₄ alkoxy; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide.

16. The complex of claim 13, wherein R¹ is C1-C4 alkyl, preferably C1-C2 alkyl, R² is _{hydrogen; preferably –O–; k is} zero or 1, 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide.

17. The complex of any one of claims 13 to 17, wherein the conjugate corresponds in structure to: , , , or wherein each of Y¹ and Y³ is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH).

18. A kit comprising: (i) an anti-nitazene antibody; and (ii) a conjugate corresponding in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase; and optionally, wherein when R¹ is hydrogen or CH₂CH₃ and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or –O(CH2)3- CH₃.

19. The kit of claim 18, wherein the conjugate corresponds in structure a Formula (Ic): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g and k are each zero or 1; h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase; and optionally, wherein when R¹ is hydrogen or CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or –O(CH₂)_3- CH3.

20. The kit of claim 18 or claim 19, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).

21. The kit of claim any one of claims 18 to 20, wherein the anti-nitazene antibody is raised against the complex of claims 1-5 and 12-17 wherein the immunogenic carrier is present.

22. A method of detecting the presence of nitazene and/or a metabolite of nitazene in a sample, the method comprising the steps of: combining the sample, a conjugate, and an anti-nitazene antibody in a medium; and examining the medium for the presence of a complex comprising nitazene and the anti- nitazene antibody, wherein the conjugate corresponds in structure to a Formula (Ib): wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , g, i, and k are each zero or 1; h, j, and l are each zero to 100; and each of Y¹, Y², and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase; and optionally, wherein when R¹ is hydrogen or –CH₂CH₃ and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH3, –OCH2CH3, –O(CH2)2CH3, –OCH(CH3)2, or – O(CH₂)₃CH₃.

23. The method of claim 22, wherein the conjugate corresponds in structure a Formula (Ic): (Ic) wherein: R¹, R², and R³ are each independently hydrogen, an alkyl, an alkoxy, , or –NH–; g are zero or h and l are each zero to 100; and each of Y¹ and Y³ is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase; and optionally, wherein when R¹ is hydrogen or –CH2CH3 and R² is hydrogen, then R³ is not hydrogen, –OH, –OCH₃, –OCH₂CH_3, –O(CH₂)₂CH_3, –OCH(CH₃)₂, or – O(CH2)3CH3.

24. The method of claim 22 or claim 23, wherein the anti-nitazene antibody is raised against the complex of claims 1-5 and 12-17 wherein the immunogenic carrier is present and/or the sample is one or more selected from the group consisting of urine, whole blood, plasma, serum, lymph, mucus, expressed breast milk, semen, stool, sputum, cerebral spinal fluid, tears, hair, saliva, cells, tissues, an organ, and/or a biopsy.

25. A method of preparing an antibody, the method comprising: immunizing an antibody-producing animal with an immunogen comprising a composition comprising the complex of anyone of claims 1-17; and isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen.