Xylazine haptens, immunogens, and protein conjugates and methods of production and use thereof

Abstract

Compositions, kits, and systems containing xylazine haptens, immunogens, and / or protein conjugates are disclosed. Also disclosed are methods of producing and using the xylazine haptens, immunogens, and conjugates.

Description

XYLAZINE HAPTENS, IMMUNOGENS, AND PROTEIN CONJUGATES AND METHODS OF PRODUCTION AND USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 589,977, filed October 12, 2023. The entire contents of the abovereferenced patent application(s) are hereby expressly incorporated herein by reference.BACKGROUND

[0002] Xylazine is a drug used for sedation, anesthesia, muscle relaxation, and analgesia in animals such as horses, cattle, and other non-human mammals. It is an analog of clonidine and an agonist at the a? class of adrenergic receptor. Xylazine has become a commonly used non-prescribed drug in the United States, where it is known by the street name "tranq." particularly in Puerto Rico. Fentanyl mixed with xylazine is known by the street names "sleep-cut," "zombie drug," and "tranq dope." Xylazine is also frequently found in "speedball," which is a mixture of several drugs that typically include cocaine, heroin or morphine, and fentanyl. From November 2021 until August 2022, 80% of drug paraphernalia which tested positive for fentanyl at needle exchange programs in Maryland also contained xylazine.

[0003] Development of monoclonal antibodies to xylazine have been described in Chao et al. (New Journal of Chemistry (2021) 45:4658-4665), Wei et al. (CN116003616A), and Xu et al. (CN111454912A). However, while the number of drug abuse cases of xylazine has increased exponentially, there is currently no commercially available diagnostic immunoassay for detection of xylazine in biological samples.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 contains chemical structures of various non-limiting embodiments of xylazine haptens constructed in accordance with the present disclosure, wherein the haptens contain linkers at different positions.

[0005] FIG. 2 contains chemical structures of various non-limiting embodiments of xylazine immunogens and protein conjugates constructed in accordance with the presentdisclosure. Non-limiting examples of "Protein" that may be conjugated to xylazine as shown include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), glucose-6-phosphate dehydrogenase (G6PDH), any carrier or label peptides or proteins, and any combinations thereof.

[0006] FIG. 3 contains a scheme for synthesis of certain non-limiting embodiments of XLZ1 haptens for immunogen synthesis and conjugates in accordance with the present disclosure. The labels (l)-(10) shown in FIG. 3 equate to the Formulas disclosed herein as follows: (1) Formula XI; (2) Formula II; (3) Formula XII; (4) Formula XIII; (5) Formula XIV; (6) Formula XV; (7) Formula XVI; (8) Formula XVII; (9) Formula XVIII; and (10) Formula XIX.

[0007] FIG. 4 contains another scheme for synthesis of certain non-limiting embodiments of XLZ1 immunogens and conjugates in accordance with the present disclosure. Non-limiting examples of "Proteins" that may be conjugated thereto include KLH, BSA, OVA, G6PDH, and / or any carrier and / or label proteins. The labels (3), (6), and (10) shown in FIG. 4 equate to the Formulas disclosed herein as follows: (3) Formula XII; (6) Formula XV; and (10) Formula XIX.

[0008] FIG. 5 contains a scheme for synthesis of particular (but non-limiting) embodiments of xylazine conjugates, XLZ1-C6-3K G6PDH and XLZ1-C11-3K G6PDH conjugates. The Formulas disclosed therein are as follows: Upper Left structure, Formula XVII; Upper Middle structure, Formula XXII; Upper Right structure, Formula XXIII; Lower Left structure, Formula XVII; Lower Middle structure, Formula XXIX; and Lower Right structure, Formula XXX.

[0009] FIG. 6 contains a scheme for synthesis of a particular (but non-limiting) embodiment of a xylazine hapten, XLZ2-O4-C2S2 hapten.

[0010] FIG. 7 contains another scheme for synthesis of certain non-limiting embodiments of XLZ2-O4-C2SAc-immunogens and conjugates in accordance with the present disclosure. Non-limiting examples of "Proteins" that may be conjugated thereto include KLH, BSA, OVA, G6PDH, and / or any carrier and / or label proteins. The Formulas disclosed on the right side of the figure are as follows: Top structure, Formula XXVI; Middle structure, Formula XXVII; Bottom structure, Formula XXVIII (where R4 is G6PDH).

[0011] FIG. 8 contains yet another scheme for synthesis of certain non-limiting embodiments of XLZ2-O4-C8-Br and XLZ2-O4-C6-Br haptens in accordance with the present disclosure.

[0012] FIG. 9 contains yet another scheme for synthesis of certain non-limiting embodiments of XLZ2-O4-C6-3K G6PDH and XLZ2-O4-C8-3K G6PDH conjugates in accordance with the present disclosure.

[0013] FIG. 10 contains a further scheme for synthesis of certain non-limiting embodiments of xylazine haptens / immunogens / conjugates in accordance with the present disclosure. Non-limiting examples of "Proteins" that may be conjugated thereto include KLH, BSA, OVA, G6PDH, and / or any carrier and / or label proteins.

[0014] FIG. 11 contains yet another scheme for synthesis of certain non-limiting embodiments of xylazine haptens / immunogens / conjugates in accordance with the present disclosure. Non-limiting examples of "Proteins" that may be conjugated thereto include KLH, BSA, OVA, G6PDH, and / or any carrier and / or label proteins.

[0015] FIG. 12 contains yet another scheme for synthesis of certain non-limiting embodiments of xylazine haptens / immunogens / conjugates in accordance with the present disclosure.DETAILED DESCRIPTION

[0016] 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.

[0017] 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 connectionwith, 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.

[0018] 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.

[0019] 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.

[0020] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0021] 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."

[0022] 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.

[0023] 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).

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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, alinear, 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.

[0031] The term "hydrocarbyl" refers to an organic group that consists solely of carbon and hydrogen. A hydrocarbyl group may be unsaturated or it may contain one or more carbon-carbon double bonds or one or more carbon-carbon triple bonds or a mixture thereof. The term "hydrocarbyl" includes alkyl, alkenyl and alkynyl.

[0032] The phrase "bulky organic" refers to an organic group that exhibits a large molecular size for its weight. The bulky organic hinders the ability of a specific binding member to bind to an area of a molecule that comprises the bulky organic group. The phrase "bulky hydrocarbyl" refers to a hydrocarbyl group that exhibits a large molecular size for its weight such as, for example, that exhibited by an alkyl group that is branched or cyclic.

[0033] The phrase "non-bulky organic" refers to an organic group that does not exhibit a large molecular size for its weight. The non-bulky organic group does not hinder to a significant degree the ability of an antibody to bind to an area of a molecule that comprises the non-bulky organic group. The phrase "non-bulky hydrocarbyl" refers to a hydrocarbyl group that does not exhibit a large molecular size for its weight such as that exhibited by a straight chain alkyl group.

[0034] 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 3to 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.

[0035] Bulky hydrocarbyl includes branched chain hydrocarbyl and cyclic hydrocarbyl. Bulky branched chain hydrocarbyl has branching at or near the carbon atom that is attached to another molecule. Examples of bulky branched chain alkyl include, but are not limited to, sec-butyl, tert-butyl, triethylmethyl, diethylmethyl, tripropylmethyl and dipropylmethyl, for example. Cyclic alkyl is alkyl comprising one or more rings. Examples of cyclic alkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and norbornyl, for example. Examples of non-bulky alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, for example.

[0036] The term "alkenyl" refers to a hydrocarbyl group having hydrocarbon chains of the number of carbon atoms specified above of either a straight- or branched-configuration and having at least one carbon-carbon double bond, which may occur at any point along the hydrocarbon chain, examples of which include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, dimethyl pentenyl, for example.

[0037] The term "alkynyl" refers to a hydrocarbyl group having hydrocarbon chains of the number of carbon atoms specified above containing at least one carbon-carbon triple bond, including, but not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, and 2-butynyl, for example.

[0038] The term "lower hydrocarbyloxy" refers to a hydrocarbyl group that is 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 a hydrocarbyl group to a parent compound.

[0039] The term "lower 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 to a parent compound.

[0040] As used herein, the term "aryl" refers to an organic group derived from an aromatic hydrocarbon by the removal of one atom and containing one or more aromatic rings such as, but not limited to, 1 to 5 aromatic rings, or 1 to 4 aromatic rings, or 1 to 3 aromatic rings, or 1 to 2 aromatic rings, or 2 to 4 aromatic rings, or 2 to 3 aromatic rings, for example. Examples of aryl include, but are not limited to, phenyl (from benzene), naphthyl (fromnaphthalene), and anthracyl (from anthracene), for example. The aryl group may be substituted or unsubstituted. "Substituted aryl" refers to aryl groups that comprise one or more substituents such as, but not limited to, a bulky hydrocarbyl, a non-bulky hydrocarbyl, a functional group (e.g., chloro, bromo, iodo, fluoro, nitro and sulfone), for example.

[0041] As used herein, "arylhydrocarbyl" refers to an organic group having a lower hydrocarbyl group to which is attached an aryl group. As used herein, "aralkyl" refers to an organic group having a lower alkyl group to which is attached an aryl group such as, but not limited to, benzyl, phenethyl, 3-phenylpropyl and 1-naphthylethyl, for example.

[0042] 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.

[0043] 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.

[0044] 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')z, 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 thatretain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, e.g., Hudson et al. (Nature Med. (2003) 9:129-134). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).

[0045] 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 "antigenbinding 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.

[0046] 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.

[0047] 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.

[0048] 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 crossspecies 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).

[0049] 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.

[0050] 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.

[0051] 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 IO-6M, IO-7M, IO-8M or IO-9M. When an antibody specifically binds to a receptor or ligand (i.e., counterreceptor), it may substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when an excess of antibody reduces the quantity of receptor bound to ligand by at least about 20%, 40%, 60% or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0052] 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 therapeuticuses 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 crossreactivity to other, related antigens.

[0053] 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%.

[0054] 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 monoclonalantibodies produced in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0055] 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.

[0056] 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.

[0057] An "analyte" is a molecule that is capable of being recognized by an analytespecific 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).

[0058] Turning now to the inventive concepts, certain non-limiting embodiments of the present disclosure include xylazine haptens, immunogens, and protein conjugates, as well as compositions, kits, and systems including same and methods of producing and using same.

[0059] Certain non-limiting embodiments of the present disclosure are directed to a composition comprising a compound having the structure of Formula I:with the proviso that at least one of Ri, R2, and R3 is not H. In the native xylazine molecule, each of Ri, R2, and R3 is H.

[0060] In certain particular (but non-limiting) embodiments, the at least one of Ri, R2, and R3 that is not H comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non- bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyloxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

[0061] In certain particular (but non-limiting) embodiments, the compound comprises the structure of one of Formulas II, III, IV, V, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XXII, or XXIX:ormu aFormula XHI Formula XIV Formula XVFormula XVIII Formula XIX Formula XXIIFormula XXIX

[0062] In other particular (but non-limiting) embodiments, the compound has the structure of one of Formulas VI, VII, VIII, IX, XX, XXI, XXIII, or XXX:Formula XXX wherein R4 is a peptide or protein. Non-limiting examples of peptides or proteins that may be utilized in accordance with the present disclosure include carrier and label proteins. In a particular (but non-limiting) embodiment, R4 is an immunogenic carrier.

[0063] Any immunogenic carriers known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Non-limiting examples thereof include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, a polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), a carrier protein, a label protein, and combinations thereof.

[0064] Certain non-limiting embodiments of the present disclosure are directed to a composition comprising a compound having the structure of Formula X:wherein one of Rs and Re is present and comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non-bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyl oxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

[0065] In certain particular (but non-limiting) embodiments, the compound has the structure of one of Formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI:Formula XXVFormula XXVHIFormula XXXII Formula XXXIVFormula XXXVI wherein R4 is a peptide or protein.

[0066] In certain particular (but non-limiting) embodiments, R5 or Re comprises an immunogenic carrier, such as (but not limited to) any of the immunogenic carriers disclosed or otherwise contemplated herein.

[0067] The compositions of the present disclosure may include one or more additional ingredients, such as (but not limited to) at least one excipient.

[0068] Certain non-limiting embodiments of the present disclosure are directed to methods of preparing any of the compositions disclosed or otherwise contemplated herein. In certain particular (but non-limiting) embodiments, the methods include one or more steps as illustrated in FIGS. 3-9. For example (but not by way of limitation), the method may include the step of attaching a tert-butoxycarbonyl (Boc) protecting group to the amine of the xylazine molecule to direct attachment of the various R group(s) to the position(s) shown rather than to the amine group.

[0069] Certain non-limiting embodiments of the present disclosure are directed to a method of preparing an antibody. In the method, an antibody-producing animal is immunized with an immunogen comprising any of the compositions disclosed or otherwise contemplated herein, and antibodies that specifically bind to the immunogen are isolated from the animal.

[0070] The immunization and isolation steps may be performed by any methods known in the art or otherwise contemplated herein. In certain particular (but non-limiting) embodiments, the step of isolating antibodies further comprises the steps of: isolating antibody-producing spleen cells from the antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to the immunogen; and preparing a hybrid cell line by fusing the selected antibody-producing spleen cell(s) from the antibody-producing animal with an appropriate fusion partner, whereby the hybrid cell line secretes the monoclonal antibody.

[0071] Certain non-limiting embodiments of the present disclosure are directed to at least one antibody produced by the methods disclosed or otherwise contemplated herein.

[0072] Certain non-limiting embodiments of the present disclosure are directed to a conjugate composition that comprises any of the compositions disclosed or otherwise contemplated herein and having an enzyme coupled to the composition. Any enzymes known in the art or otherwise contemplated herein for use in diagnostic assay systems may be utilized in accordance with the present disclosure. Non-limiting examples of enzymes that may be utilized include glucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase, P-galactosidase, and horseradish peroxidase.

[0073] Certain non-limiting embodiments of the present disclosure are directed to a kit that comprises at least one of any of the haptens, immunogens, conjugates, and / or antibodies disclosed or otherwise contemplated herein. A particular (but non-limiting) embodiment includes a kit that comprises at least one antibody and at least one conjugate.

[0074] Certain non-limiting embodiments of the present disclosure are directed to a method of detecting xylazine in a sample. In the method, a sample suspected of containing xylazine is combined with any of the antibodies disclosed or otherwise contemplated herein, and the presence of xylazine in the sample is detected based on the specific binding of the antibody thereto.

[0075] Certain non-limiting embodiments of the present disclosure are directed to a method of detecting xylazine in a sample. In the method, a sample suspected of containing xylazine is combined with one or more components of any of the kits disclosed or otherwise contemplated herein, and the presence of an enzymatic reaction product of the conjugate is detected. It is determined that xylazine is present in the sample when a decrease in the enzymatic reaction product of the conjugate is observed when compared to enzymatic reaction product detected in the absence of sample.

[0076] 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 xylazine are disclosed in US Patent No. 7,022,492 (the entirety of which is hereby expressly incorporated herein by reference).EXAMPLES

[0077] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.Example 1: General Synthesis Procedures

[0078] Materials and Equipment: The compounds were purified on a Shimadzu HPLC system (Riverwood, MD) equipped with a Silica-bond - C18 reverse phase column and Biotage LC (Charlotte, NC). The chemical reactions were monitored by TLC (thin layer chromatography) using Silica gel plates from Analtech 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), Fluka (Waltham, MA), ThermoScientific (Waltham, MA), VWR (Radnor, PA) and used as received.TH NMR was recorded on a Bruker UltraShield™ 600 MHz spectrometer (Bruker, Billerica, MA). Chemical shifts were reported in parts per million (ppm, 6) and related to tetramethylsilane or with deuterated solvent as internal reference. NMR abbreviations used are: s (singlet), brs (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.

[0079] The following abbreviations have the meanings set forth below:

[0080] ACN - acetonitrile

[0081] AcOH - acetic acid

[0082] BoczO - di-tert-butyl dicarbonate

[0083] BrAcSu - bromoacetic acid N-hydroxysuccinimide ester

[0084] BSA - bovine serum albumin

[0085] CA - cellulose acetate filters

[0086] calcd. - calculated

[0087] CDCN-ds - deuterated Acetonitrile having 3 deuterium atoms (for NMR spectra)

[0088] CDCh - deuterated Chloroform (for NMR spectra)

[0089] CV - column volume

[0090] DCM - dichloromethane

[0091] DI water - deionized water

[0092] DIPEA - N,N-Diisopropylethylamine

[0093] DMF - N,N-dimethylforrnarnide

[0094] DTT - Dithiothreitol

[0095] EDC or EDC-HCI - N-(3-dimethylarninopropyl)-N'-ethylcarbodiimide hydrochloride

[0096] eq. - molar equivalent

[0097] EDTA - ethylenediaminetetraacetic acid

[0098] EtOAc - ethyl acetate

[0099] g - grams

[0100] G6PDH - glucose-6-phosphate native enzyme

[0101] G6PDNa2 - glucose-6-phosphate disodium salt (substrate)

[0102] GMBS - N-y-maleimidobutyryl-oxysuccinimide ester

[0103] h - hour

[0104] 1H - proton (for NMR)

[0105] HPLC - high-performance liquid chromatography

[0106] KLH - keyhole limpet hemocyanin

[0107] LC - liquid chromatography

[0108] min - minute

[0109] MeOH - methanol

[0110] MeOD-ck - deuterated methanol having 4 deuterium atoms (for NMR spectra)

[0111] mg - milligram

[0112] pg - microgram

[0113] pL- microliter

[0114] MHz - megahertz

[0115] mmol, mM - millimole

[0116] MWCO - molecular weight cut-off

[0117] mL - milliliter

[0118] NaOAc - sodium acetate

[0119] P-NADH - nicotinamide adenine dinucleotide

[0120] NEts - triethylamine

[0121] G6PDH - native glucose-6-phosphate dehydrogenase enzyme

[0122] NMR - nuclear magnetic resonance

[0123] OVA - ovalbumin isolated from chicken egg white (Sigma)

[0124] PB - Phosphate buffer

[0125] RPM - rotations per minute

[0126] rt - room temperature

[0127] 3K G6PDH - recombinant G6PDH enzyme

[0128] SuOH -N-hydroxysuccinimide

[0129] TCEP-HCI - tris(2-carboxyethyl)phosphine hydrochloride

[0130] TFA - trifluoroacetic acid

[0131] TRIS - tris(hydroxymethyl)aminomethane

[0132] UV - ultraviolet

[0133] v / v - volume ratio

[0134] XLZ1- xylazine first series of conjugates / immunogens

[0135] XLZ2- xylazine second series of conjugates / immunogensExample 2: Experimental XLZ 1 Hapten Syntheses for Immunogens and Native G6PDH Conjugates (FIG. 3)

[0136] Preparation of XLZl-C4-OtBu (1): In a vial equipped with a magnetic stir bar and a cap, xylazine (95 mg, 0.43 mmol) was dissolved in DMF (500 pL); then, K2CO3 (120 mg, 0.87 mmol, 2 eq.) and tert-butyl 4-iodobutanoate (0.65 mmol, 1.5 eq., 142 pL) were added. The resulting reaction mixture was heated at 50 -70°C for 6 days and 90% of the xylazine reacted. The reaction was continued until completion (an additional 3 days). The product was purified by Biotage LC using DCM / MeOH 70 / 30 v / v. Fractions containing the product were pooled out concentrated in vacuum to give 123 mg (0.34 mmol) of oily product in 80% yield. Ms. calcd. for [C2oH3iN202S+] 363.21, found 363.33.

[0137] XH NMR (600 MHz, CDCI3) 7.12 (t, J = 7.86 Hz, 1H), 7.05 (d, J = 7.60 Hz, 2H), 3.66 - 3.64 (m, 2H), 3.60 - 3.57 (m, 2H), 2.89 - 2.87 (m, 2H), 2.23 (s, 6H), 2.20 - 2.19 (m, 2H), 1.89 - 1.76 (m, 4H), 1.43 (m, 2H), 1.37 (s, 9H).

[0138] Preparation of XLZ1-C4-OH (2): In a vial equipped with a magnetic stir bar, XLZ1- C4-OtBu (80 mg, 0.22 mmol) was dissolved in DCM (200 pL) then TFA (200 pL) was added. The resulting reaction mixture was stirred at room temperature (rt.) for 5 days. The solvent wasevaporated on a rotatory evaporator to give an oil which was further dried on an oil pump.  MS Calcd. for [C16H23N2O2S+] 307.15, found 307.30.

[0139] Preparation of XLZ1‐C4‐OSu (3): In a vial equipped with a magnetic stir bar and a  cap, XLZ1‐C4‐OH (2) (7.8 mg, 0.025 mmol) was dissolved in DMF (156 µL), then EDC*HCl (6  mg 0.031 mmol) and SuOH (6 mg, 0.052 mmol) were added. The resulting reaction mixture was stirred at  rt.  for  two days and monitored by UHPLC MS. MS calcd.  for  [C20H26N3O4S+] 404.16, found 404.32. Once the OSu activation was completed, DMF (0.156 mL) was added  to make a 25 mg / mL XLZ1‐C4OSu (3)‐DMF solution. This solution was used for the immunogen  synthesis.

[0140] Preparation of XLZ1‐C5‐OtBu (4): In a vial equipped with a magnetic stir bar and a  cap, xylazine (130 mg, 0.59 mmol) was dissolved in DMF (500 µL), then K2CO3 160 mg (1.18  mmol, 2 eq.) was added followed by tert‐Butyl 5‐bromovalerate (209 mg, 1.5 eq., 160 µL) and  KI (~50 mg). The resulting reaction mixture was heated at 100‐120 0C for 6 days and 90% of  the xylazine reacted. The product was purified by reverse phase, lyophilized overnight, to give  180 mg (0.48 mmol) of product (4) in 81% yield.

[0141] Mobile phase: Solvent A: Water + 0.1% AcOH; Solvent B ACN + 0.1% AcOH; LC  purification Program: 0.00 – 1.00 min isocratic Solvent B at 10%, 1.00 – 24.05 min gradient  Solvent B to 80%, 24.05‐28.00 isocratic Solvent B at 80%, 28.00 – 32.00 min isocratic Solvent  B to 10%, 32.05 min collection stopped. Fractions collected between 12 – 15 mins contained  the desired product.

[0142] Ms. calcd for [C20H33N2O2S+] 377.23, found 377.38. 1H NMR (600 MHz, CDCl3) 7.14  – 7.11 (m, 1H), 7.05 (d, J = 7.61 Hz, 2H), 3.66 – 3.63 (m, 2H), 3.56 – 3.53 (m, 2H), 2.89 – 2.87  (m, 2H), 2.23 (s, 6H), 2.18 (t, J = 6.96 Hz, 2H), 1.79 – 1.75 (m, 2H), 1.53 – 1.50 (m, 2H), 1.45 –  1.44 (m, 2H), 1.39 (s, 9H).

[0143] Preparation of XLZ1‐C5‐OH (5): In a vial equipped with a magnetic stir bar and a cap, XLZ1‐C5‐OtBu (180 mg, 0.48 mmol) was dissolved  in DCM (300 µL), then TFA (300 µL) was added. The resulting reaction mixture was stirred at rt for 7 days, and when completed  by LC‐MS the solvent was evaporated to dryness to give 152 mg of XLZ1‐C5‐OH (5) (99% yield).  MS Calcd. for [C16H25N2O2S+] 321.16 found 321.26. 1H NMR (600 MHz, CDCl3) 10. 28 (s, 1H),  7.28 (t, J = 7.71 Hz, 1H), 7.16 (d, J = 7.57 Hz, 2H), 3.78‐3.76 (m, 2H), 3.71 (t, J = 5.35 Hz, 2H),  3.04 (t, J = 6.02 Hz, 2H), 2.37 (t, J = 6.25 Hz, 2H), 2.20 (s, 6H), 2.17 – 2.16 (m, 2H), 1.62 – 1.61  (m, 4H).

[0144] Preparation of XLZ1‐C5‐OSu (6): In a vial equipped with a magnetic stir bar and a  cap, XLZ1‐C5‐OH (12 mg, 0.037 mmol) was dissolved in DMF (0.240 mL), then EDC*HCl (9 mg  0.047 mmol) and SuOH (9 mg, 0.078 mmol) were added. The resulting reaction mixtures were  stirred at rt. for two days and the reaction was monitored by LC MS. The reaction completes after 8h. MS  calcd  for  [C21H28N3O4S+] 418.18,  found 418.35. Once  the OSu activation was completed, DMF (0.240 mL) was added to make a 25 mg / mL of XLZ1‐C5‐OSu (6)‐DMF solution.  This solution was used for the preparation of immunogens and G6PDH conjugates.

[0145] Preparation of XLZ1‐C3‐NHBoc (7): In a vial equipped with a magnetic stir bar and  a cap, xylazine (140 mg, 0.64 mmol) was dissolved in DMF (750 µL), then, K2CO3 (175 mg, 1.27  mmol, 2 eq.), 3‐(amino)propyl bromide (182 mg,0.77 mmol, 1.2 eq.) and KI (100 mg) were added.  The  resulting  reaction mixture was  heated  to  120°C  for  7  days.  The  solvent was removed on a rotatory evaporator, the crude was suspended in EtOAc (20 mL) and washed with DI water  (2 x10 mL). The organic  layer was collected, concentrated, and dissolved  in Acetonitrile (2 mL) and purified on a Shimadzu LC equipped with a C18 column.

[0146] Mobile Phase: Solvent A Water + 0.1% AcOH, Solvent B Acetonitrile + 0.1% AcOH,  detection at 250 nm; Flowrate 20 mL / min; LC purification program: 0 – 1 min isocratic Solvent  B 20%, 1 – 20 min gradient Solvent B 60%, 20 – 24 min isocratic Solvent B 60%, 24 – 26 min  gradient Solvent B 20% 26– 28 min isocratic Solvent B 20%, 28.01 min controller stopped.

[0147] Fractions containing the product were collected, concentrated, and lyophilized to  give 160 mg of product (7) (120 mg of the main peak and 40 mg of the peak tail). MS Calcd.  for [C20H32N3O2S+] 378.22, found 378.38.

[0148] 1H NMR (600 MHz, CDCl3): 8.14 (brs, 2H), 7.22 (t, J = 7.62 Hz, 1H), 7.10 (d, J = 7.58  Hz, 2H), 3.77 (t, J = 7.71 Hz, 2H), 3.73 (t, J = 5.26 Hz, 2H), 3.13 (brs, 2H), 2.95 (t, J = 5.90 Hz,  2H), 2.21 (s, 6H), 2.06 (s, 3H), 1.98 (s, 2H), 1.76 (t, J = 6.77 Hz, 2H), 1.43 (s, 9H).

[0149] Synthesis of XLZ1‐C3‐NH2 (8):  In a vial equipped with a magnetic stir bar and a cap,  XLZ1‐C3NHBoc  (7)  (40 mg)  and DCM / TFA 1 / 1  v / v  300 µL were  added.  The  resulting reaction mixture was stirred for 3 days. Solvent was removed on a rotatory evaporator, then  lyophilized to give 30 mg of colorless oil of XLZ1‐C3NH2 TFA salt (8). MS Calcd. for [C15H24N3S+]  278.44, found 278.25.

[0150] Preparation of XLZ1‐C8‐OH (9): In a vial equipped with a magnetic stir bar and a  cap, XLZ1‐C3NH2 TFA salt (8) (30 mg, 0.076 mmol) was dissolved in THF (0.2 mL), DIPEA (20 µL) was  added  followed by  succinic  anhydride  (8 mg, 0.08 mmol).  The  resulting  reaction mixture was stirred at rt. overnight, concentrated then dissolved  in ACN and purified on a reverse phase HPLC equipped with a C18 column. Fractions containing the product, XLZ1‐C8‐ OH (9), were collected, concentrated, and lyophilized (33.5 mg). Ms. calcd. for [C19H28N3O3S+]  378.19, found 378.30.

[0151] Mobile phase: Solvent A Water + 0.05% TFA, Solvent B Acetonitrile + 0.05% TFA;  detection at 250 nm; Flowrate 7 mL / min; LC purification program: 0 – 2 min isocratic Solvent  B 20%, 2 – 14 min gradient Solvent B 70%, 14 – 16 min isocratic Solvent B 70%, 16 – 17 min  gradient Solvent B 20% 17 min – 21 min isocratic Solvent B 20%.

[0152] 1H NMR (600 MHz, CDCl3): 9.94 (s, 1H), 8.51 (s, 2H), 7.66 (s, 1H), 7.29 (t, J = 7.65  Hz, 1H), 7.16 (d, J = 7.60 Hz, 2H), 3.78 (t, J = 7.46 Hz, 2H), 3.73 (brs, 2H), 3.26 (s, 2H), 3.05 (t, J  = 5.55 Hz, 2H), 2.66 (brs, 2H), 2.55 (brs, 2H), 2.19 (m, 8H), 1.75 (m, 2H). After lyophilization,  22.5 mg of product was recovered as a white powder.

[0153] Preparation of XLZ1‐C8‐OSu (10): In a glass vial equipped with a magnetic stir bar  and a cap, XLZ1‐C8‐OH (9) (11 mg) was dissolved in DMF (200 µL) then EDC (9 mg) and SuOH  (9 mg) were added. The resulting reaction mixture was stirred for 3 days, then additional EDC  (9 mg), SuOH (6 mg) were added. The resulting reaction mixture was stirred until completed  by UPLC ‐MS to give the product (10). MS Calcd. for [C23H31N4O5S+] 475.58, found 475.40. DMF  (240 µL) was then added to make a 25 mg / mL hapten‐DMF solution.    Example 3: Bioconjugation

[0154] Bioconjugation buffers

[0155] Buffer 1: 50 mM Phosphate buffer, pH 8.00; Buffer 2: 100 mM Phosphate buffer,  pH 8.00; Buffer 3: 55 mM TRIS buffer, pH 8.00; Buffer 4: 50 mM Phosphate buffer, pH 7.00;  Buffer 5: 55 mM TRIS buffer, pH 7.00; Buffer 6 50 mM Phosphate buffer, 1mM EDTA, pH 7.25;  Buffer 7: 50 mM Phosphate buffer, 1mM EDTA, pH 7.25, 0.025 mM DTT; Buffer 8: 50 mM  Phosphate buffer pH 7.5.

[0156] Preparation of XLZ1‐C4‐OVA conjugate (11a): In a plastic tube, OVA (10 mg) was  suspended in buffer 1 (2 ml) then, XLZ1‐C4‐OSu (3) (1.9 mg, 75 µL, corresponding to 20x mol  excess to MW of 42,700 Da) was added dropwise; the resulting reaction mixture was rocked  at rt. for 16h, then purified on G25M column (CV=60 mL) pre‐equilibrated with buffer 4. A  volume of 7 mL of conjugate solution was collected which was then concentrated on Amicon  stir cell MW CO 10kDa to 4.0 mL, conc = 0.72 mg / mL.

[0157] Bioconjugation Process for XLZ1-C4 Immunogens (FIG. 4)

[0158] Preparation of XLZ1-C4-BSA immunogen (lib): In a glass vial equipped with a magnetic stir bar and a cap, BSA (20 mg) was suspended in buffer 1 (3 ml) then, XLZl-C4-OSu (3) (2.34 mg, 94 pL, corresponding to 20x mol excess to MW of 69,000 Da) was added dropwise; the resulting reaction mixture was stirred at rt. for 16h, then purified on G25M column (CV=60 mL) preequilibrated with buffer 4. A volume of 15 mL of conjugate solution was collected which was concentrated on Amicon stir cell MW CO lOkDa to 5.0 mL (cone = 3.82 mg / mL).

[0159] Preparation of XLZ1-C4-KLH immunogen (11c): In a glass vial equipped with a magnetic stir bar and a cap, KLH (20 mg) was suspended in buffer 1 (3 ml) then, XLZl-C4-OSu (3) (2.56 mg, 102 pL, corresponding to 200x mol excess to MW average of 635,000 Da) was added dropwise; the resulting reaction mixture was stirred at rt. for 16h, then purified on G25M column (CV=60 mL) preequilibrated with buffer 4. A volume of 15 mL of conjugate solution was collected which was concentrated on Amicon stir cell MW CO lOkDa to 3.8 mL, (cone = 5.0 mg / mL).

[0160] Bioconjugation process - for XLZl-C5-immunogens (FIG. 4)

[0161] Preparation of XLZ1-C5-OVA conjugate (12a): In a glass vial equipped with a magnetic stir bar and a cap, OVA (20 mg) was suspended in buffer 2 (3 ml), then XLZl-C5-OSu (2.71 mg, 108 pL, corresponding to 20x mol excess to MW of 42,700 Da) was added dropwise. The resulting reaction mixture was stirred at rt. for 16h, then purified on G25M column (CV = 60 mL) preequilibrated with buffer 4. A volume of 15 mL of conjugate solution was collected, which was concentrated on Amicon stir cell MW CO lOkDa to 7.0 mL (cone = 2.97 mg / mL).

[0162] Preparation of XLZ1-C5-BSA immunogen (12b): In a glass vial equipped with a magnetic stir bar and a cap, BSA (20 mg) was suspended in buffer 2 (3 ml) then, XLZl-C5-OSu (3.72 mg, 148 pL, corresponding to 40x mol excess to MW of 69,000 Da) was added dropwise; the resulting reaction mixture was rocked at rt. for 16h, then purified on G25M column (CV= 55 mL) pre-equilibrated with buffer 4. A volume of 15 mL of conjugate solution was collected which was concentrated on Amicon stir cell MW CO lOkDa to 5.2 mL (cone = 4.0 mg / mL).

[0163] Preparation of XLZ1-C5-KLH immunogen (12c): In a glass vial equipped with a magnetic stir bar and a cap, KLH (20 mg) was suspended in buffer 2 (3 ml) then, XLZl-C5-OSu (2.02 mg, 81 pL, corresponding to 200x mol excess to MW average of 635,000 Da) was added dropwise; the resulting reaction mixture was rocked at rt. for 16h, then purified on G25Mcolumn (CV = 55 mL) preequilibrated with buffer 4. A volume of 18 mL of conjugate solution was collected which was then concentrated on Amicon stir cell to 4.0 mL (cone = 5.0 mg / mL).

[0164] Preparation of XLZ1-C5- native G6PDH conjugates

[0165] The G6PDH enzyme (24 mg, ~ 2 mL) was centrifuged at 9.0 g for 30 mins at 4 °C on a bench-top centrifuge; the resulting supernatant was disposed, the bottom white cake was then dissolved in buffer 3 (3 mL). The resulting clear solution was then loaded onto a dialysis bag and buffer exchanged with buffer 3 (3 x 500 mL) in a cold room. After dialysis, 3 mL of enzyme solution was recovered; the concentration was measured at 40x dilution and found to be 7.5 mg / mL. The concentration was then adjusted to 5 mg / mL by adding buffer 3 (1.5 mL); the concentration was measured again at 40x dilution, and it was found to be 5.1 mg / mL.

[0166] The enzyme solution was chilled on ice bath, then G6PNa2 (30 mg) was added, the resulting reaction mixture was gentle mixed until salt dissolved, then P-NADH (40 mg) was added. The solution was mixed until all solids were dissolved.

[0167] The G6PDH enzyme (0.98 mL, 5 mg) was placed in 4 tubes, then XLZl-C5-OSu hapten (6) (at 25 mg / mL in DMF) was added to each tube at 5x, lOx, 15x, 20x mol excess. The resulting reaction mixtures were rocked at rt. (18.2 °C) for 90 mins, then quenched with L- Lysine (1.0 M) at 30x mol excess respective to the hapten. The conjugates were then purified on G50-M column (CV=60 mL) using buffer 5. A volume of 15 mL of each conjugate was collected, which was concentrated to ~3.0 mL; each conjugate had a concentration of 1.1 to 1.5 mg / mL.TABLE 1

[0168] Preparation of XLZ1-C8-OVA conjugate (14a): In a glass vial equipped with a magnetic stir bar and a cap, OVA (10 mg) was suspended in buffer 2 (2 ml) then, XLZ-C8-OSu (10) (2.23 mg, 110 pL, corresponding to 20x mol excess) was added dropwise. The resulting reaction mixture was stirred at rt. for 16h, then purified on G25M column (CV=60 mL)preequilibrated with buffer 4. A volume of 15 mL of conjugate solution was collected, which was concentrated on Amicon stir cell MW CO 10k Da to 7.0 mL, (cone = 2.5 mg / mL).

[0169] Preparation of XLZ1-C8-BSA immunogen (14b): In a glass vial equipped with a magnetic stir bar and a cap, BSA (20 mg) was dissolved in buffer 2 (3 mL) to make a 7 mg / mL solution. The resulting mixture was cooled to 4°C on an ice bath, then the hapten-DMF solution (160 pL, 4.01 mg, 25x mol excess) was added dropwise. The resulting rection mixture was stirred at rt. for 16h then purified on a G25M -Sephadex (CV = 55 mL) column using buffer 4. A volume of 14 mL of conjugate solution was collected which was concentrated on Amicon stirring cell (MW CO 10,000 Da) to 5.0 mL (cone = 3.71 mg / mL).

[0170] Preparation of XLZ1-C8-KLH immunogen (14c): In a glass vial equipped with a magnetic stir bar and a cap, KLH (20 mg) was dissolved in buffer 2 (3 mL) to make a 7 mg / mL solution. The resulting mixture was cooled to 4°C on an ice bath, then the hapten-DMF solution (160 pL, 4.01 mg) was added dropwise. The resulting rection mixture was stirred at rt. for 16h then purified on a G25M -Sephadex (CV = 70 mL) column using buffer 4. A volume of 14 mL of conjugate solution was collected which was concentrated on Amicon stirring cell (MW CO 10,000 Da) to 5.0 mL (cone = 4.0 mg / mL).

[0171] Preparation of XLZ1-C8- native G6PDH conjugates (15a-d):

[0172] G6PDH enzyme (3.8 mL, 40 mg) was centrifuged at 9.0 g for 30 mins at 4°C on a bench-top centrifuge; the resulting supernatant solution was discarded, the bottom white cake was then dissolved in buffer 3 (5 mL). The resulting clear solution was then loaded onto a dialysis bag and buffer exchanged with buffer 3 (3 x 500 mL) in a cold room. After dialysis, 5 mL of enzyme solution was recovered; the concentration was measured at 40x dilution and was found to be 7.71 mg / mL. The concentration was then adjusted to 5 mg / mL by adding buffer 3 (1.5 mL).

[0173] The enzyme solution was chilled on ice bath, then G6PNa2 (50 mg) was added, the resulting reaction mixture was gentle mixed until all substrate was dissolved; then P-NADH (50 mg) was added. The solution was mixed until all solids dissolved.

[0174] The G6PDH enzyme solution (5 mg) was placed in 4 tubes, then XLZl-C8-OSu hapten (10) (as a 20 mg / mL hapten in DMF solution) was added to each tube at 5x, lOx, 15x, 20x mol excess. The resulting reaction mixtures were rocked at rt. (18.2°C) for 90 mins, then quenched with L-Lysine (1.0 M) at 30x mol excess respective to the hapten. The conjugateswere then purified on G25-M column (CV = 60 mL) using buffer 5. A volume of ~13 mL of each conjugate was collected (Table 2).TABLE 2Example 4: Synthesis of XLZl-C3SAc 3K G6PDH conjugates (FIG. 5)

[0175] Preparation of XLZl-C3-AcBr hapten (16): In a vial equipped with a magnetic stir bar and a cap, XLZ-C3-NH2 (8) (40 mg, 0.102 mmol) was dissolved in degassed DMF (200 pL), then NEts (30 pL, 2 eq.) was added. The resulting mixture was cooled on an ice-bath, then a solution of BrAcSu (30 mg, 1.2 eq. in 200 pL THF) was added dropwise over 2 mins. The resulting reaction mixture was allowed to warm up to rt and stirred for lh. LC-MS confirmed the formation of the product, m / z calcd for C17H25BrN3OS+ 400.09 and 398.09, found 400.09 and 398.12 (as two major Br isotopes) The crude reaction mixture was injected into a Shimadzu preparative HPLC equipped with a Zorbax C18 column. The fractions containing the product were collected, concentrated, and lyophilized to give 47 mg of product 16 (0.091 mmol as TFA salt in 90% yield) as a brown oil.

[0176] Solvent A: Water +0.1 % TFA, Solvent B ACN +0.1% TFA, UV detection 254 nm.

[0177] LC purification program: 0 - 2 min isocratic solvent B 10%, 2 - 28 min gradient Solvent B 70%, 28 - 30 min isocratic Solvent B 70%, 30 - 33 min gradient Solvent B 10%, 34 min collection stopped.

[0178] Preparation of XLZ1-SC6-3K-G6PDH conjugate (17) (FIG. 5):

[0179] The 3K G6PDH enzyme (50 mg, ~5mL) was loaded onto a dialysis bag and buffer exchanged with buffer 6 (2 x 550 mL) in a cold room. The concentration was then adjusted to 5 mg / mL by adding buffer 6.

[0180] Reduction of 3K G6PDH enzyme: The 3K G6PDH enzyme (5 mg / mL, 1.1 mL) was loaded onto a plastic tube, then DTT (12 pL, as 0.5 M solution) was added. The resultingreaction mixture was blanketed with Argon, then rocked in a cold room for 16h. The next day,  it was buffer exchanged with buffer 7 on an Akta Starter equipped with G25 2 x 5mL HiTrap columns  in  a  cold  room.  Fractions  containing  the  reduced  enzyme were  collected  (1.90 mg / mL, 2 mL, 3.80 mg).

[0181] In a separate vial, XLZ1‐C3‐AcBr hapten 16 (3 mg) was dissolved in degassed DMF  (300 µL), then 0.89 µL of this solution (corresponding to 50x hapten mol excess) was added dropwise to the reduced 3K G6PDH enzyme on an  ice bath. The resulting reaction mixture was blanketed with Argon and stirred in a cold room for 16h.

[0182] The reaction was then purified on G25M column (CV = 55 mL) using buffer 4. A  fraction of 15 mL of conjugate was collected which was concentrated to 2.1 mL (1.5 mg / mL)  using an Amicon Stir cell MW CO 10,000.

[0183] Preparation of XLZ1‐C11‐MAL hapten (18): In a vial equipped with a magnetic stir  bar and a cap, XLZ‐C3‐NH2 (102 mg, 0.26 mmol, MW 391.45 as TFA salt) was dissolved in DMF  (400 µL), then NEt3 (70 µL) was added. GMBS (73 mg, 0.26 mmol, MW 280) was dissolved in DMF  (200  µL)  and  added  dropwise.  The  resulting  reaction mixture was  stirred  at  rt  and completed after 20 mins by uPLC. The reaction mixture was then diluted with ACN / Water 1 / 1  v / v 1.1 mL and injected into a Preparative LC.

[0184] Solvent A: Water + 0.1% AcOH, Solvent B Acetonitrile + 0.1% AcOH.

[0185] LC purification program: 0 – 1 min  isocratic Solvent A 10%, 1 – 20 min gradient Solvent B 80%, 20 – 24 min isocratic Solvent B 80%, 24 – 28 min gradient Solvent B 10%, 28.01  collection stopped.

[0186] The product eluted as a  sharp peak at 9 mins,  fractions 7  to 9 were collected, concentrated, and lyophilized to give 70.2 mg of product (0.159 mmol, 61% yield) as colorless  powder.

[0187] This product was placed in 5 vials: vial 1 – 15.8 mg, vial 2 – 15.3 mg, vial 3 – 15.5  mg, vial 4 – 15.1 mg, vial 5 – 8.5 mg.

[0188] MS calcd for [C23H30N4O3S+1] 443.21, found 443.68, ret. time 2.473 min.

[0189] 1H NMR (600 MHz, CDCl3‐d): 7.78 (s, 1H), 7.28 – 7.26 (m, 1H), 7.14 (d, J = 7.59 Hz,  2H), 6.66 (s, 2H), 3.92‐ 3.89 (m, 2H), 3.81 – 3.79 (m, 2H), 3.55 (t, J = 6.79 Hz, 2H), 3.27 (q, J =  5.76 Hz, 2H), 3.05 – 3.03 (m, 2H), 2.23 – 2.16 9m, 6H), 2.15 – 2.14 (m, 2H), 2.06 (s, 1H), 1.75 ‐ 1.73 (m, 2H).

[0190] Preparation of XLZ1‐C11‐3K G6PDH conjugate (19): The 3K G6PDH enzyme was  reduced following the procedure described for Conjugate 17. The 3K G6PDH enzyme (1.1 mL,  5 mg / mL) was placed into a plastic tube, then DTT (11 µL, 0.5 M) was added. The resulting  reaction mixture was blanketed with Argon and rocked in a cold room for 16h.

[0191] The next day, the reduced enzyme was buffer exchanged with buffer 7 on a G25‐ M Sephadex column G25 (CV=28 mL). A volume of 7 mL of reduced enzyme was collected  which was then concentrated on an Amicon Stir cell to approx. 1.5 mL using an Amicon Stir cell  (30,000 MW CO).  In a separate vial an XLZ‐C11‐MAL  in DMF  (10 mg / mL) solution was prepared. The enzyme was chilled on an ice bath, then 46 µL of the hapten solution was added dropwise. The  resulting  reaction mixture was blanketed with Argon and  stirred overnight (16h) in a cold room (2‐ 8°C), then purified on Sephadex G25‐M column (CV = 40 mL) using  Buffer 4. A volume of 16 mL of each conjugate was collected, which was concentrated to 2.80  mL (1.31 mg / mL) using Amicon stir cells (MW CO 30,000 cut‐off).    Example 5: Synthesis of XLZ2‐O4‐C2S2 hapten (FIG. 6)

[0192] Preparation of HO‐4‐XLZ2‐Boc (20): In a vial equipped with a magnetic stir bar and a cap, 4‐hydorxy xylazine (137 mg, 0.58 mmol) was suspended  in DMF (0.5 mL), then NEt3 (cat, 20 µL), and Boc2O / THF (290 µL, 2THF solution 1 eq.) were added. The resulting reaction  mixture was stirred for 4 hours, then diluted with EtOAc (30 mL) and washed with DI water  (2x 20 mL). The organic layer was evaporated to dryness to give 196 mg of product. MS calcd.  for [C17H24N2O3S+1] 337.16, found 280 (as major fragment, Boc‐fragmentation), 337.12. The  product was then purified by Shimadzu HPLC.

[0193] Solvent A: Water + 0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH

[0194] LC purification method: 0 – 1 min  isocratic Solvent B 10%, 1 – 20 min gradient Solvent B 80%, 20 – 24 min isocratic Solvent B 80%, 24 – 28 min gradient Solvent B 10%. The  product eluted 14‐18 mins as a broad peak, fractions containing the product were collected,  concentrated, and lyophilized to give 108 mg of compound 20 as white crystals.

[0195] 1H NMR (600 MHz, CDCl3‐d): 6.47 (s, 2H), 4.7 (broad s 1H), 3.79 (t, J = 7.2 Hz, 2H),  2.75 (t, J = 6.0 Hz, 2H), 2.07 – 2.03, m 8H), 1.48 (s, 9H).

[0196] Preparation of BocXLZ2‐O4‐C2S2 (21): In a vial equipped with a magnetic stir bar  and a cap, HO‐4‐XLZ2‐Boc (20) (85 mg, 0.253 mmol) was dissolved in DMF (0.5 mL), then K2CO3  (80 mg, 0.58 mmol) was added followed by dropwise addition of BrAcS2 linker solution (44 mg, 0.11 mmol in DMF 0.2 mL). The resulting reaction mixture was stirred at rt. for 2 days and  further heated to 60 0C for 3 days. The crude mixture was extracted with EtOAc (20 mg) and washed  with  DI  water  (2x  20  mL).  The  organic  layer  was  concentrated  on  a  rotatory evaporator and purified by LC column using Hexanes / Ethyl acetate 1:0 to 1 / 1 v / v over 15 mins  to remove the non‐polar by products. The column was further washed with EtOAc / MeOH 9 / 1  v / v (20 mL). A yellow oily compound (21) was obtained (50 mg, 0.055 mmol, 44% yield). Mass  calcd for [C42H60N6O8S4Na+] 927.33, found 927.51.

[0197] 1H NMR (600 MHz, CDCl3) 7.07 (t, J = 5.97 Hz, 2H), 6.62 (s, 4H), 4.46 (s, 4H), 3.87 –  3.83 (m, 4H), 3.66 (q, J = 6.37 Hz, 4H), 2.85 (t, J = 6.44 Hz, 4H), 2.81 (t, J = 6.68 Hz, 4H), 2.13 (s,  12H), 2.12 – 2.10 (m, 4H), 1.55 (s, 18H).

[0198] Preparation of XLZ2‐O4‐C2S2 (22):

[0199] The compound (21) was then deprotected in DCM (0.5 mL) and TFA (0.1 mL) for 4  hours and purified on a reverse phase HPLC equipped with a C18 column using Solvent A:  Water + 0.1 % AcOH and Solvent B +0.1%AcOH, monitored at 254 and 260 nm, flowrate 7.00  mL / ml.

[0200] HPLC purification program: 0 ‐ 2 min isocratic Solvent B 10%, 2 – 15 min gradient  Solvent B 70%, 15 – 17 min isocratic Solvent B 70%, 17 – 19 min gradient Solvent B 10%, 19 ‐  22 min gradient Solvent B 10%. The product eluted at 8 ‐10 mins.

[0201] After purification, 37.5 mg (0.053 mmol, 96% yield) of XLZ2‐O4‐C2‐S2 hapten (13)  was isolated as a pale‐yellow powder. Mass calcd. for [C32H44N6O4S4H+] 705.99, found 705.40  and [MNa+] 727.39., the major fragmentation 353.44 [M+2 / 2]. (2.987 and 2.98 mins)

[0202] 1H NMR (600 MHz, CD3CN‐d3) 7.34 (t, J = 5.71 Hz, 2H), 6.74 (s, 4H), 4.42 (s, 4H)  3.51 (q, J = 6.52 Hz, 4H), 3.46 – 3.44 (m, 5H), 3.04 – 3.02 (m 4H), 2.84 (t, J = 6.67Hz, 4H), 2.19  – 2.17 (m, 12H), 2.08 – 2.06 (m 4H), 1.86 (s 2H).

[0203] AcONa buffer prep. – 25 mM prepared using 75 µL of Acetic acid in 50 mL DI water  then NaOH was added to adjust the pH to 4.25‐4.30.

[0204] Hapten activation (23): In a glass vial equipped with a magnetic stir bar and a cap,  XLZ2‐O4‐C2‐S2 (22) (9 mg, 0.0127 mmol) was dissolved in EtOH (200 µL) then AcONa buffer was  added  (200  µL)  followed  by  4 mg  of  TCEP∙HCl.  The  resulting  reaction mixture was blanketed with Ar and stirred for 1h. The formation of the reduced product was confirmed by UPLC‐MS, MS Calcd.  for  [C16H24N3O2S2+] 354.13,  found 354.15  ret.  time 2.33 min  (staring material disulfide has  a  ret.  time of 2.53 min). Compound 14 was prepared  fresh before bioconjugation use.    Example 6: Bioconjugation Process for   XLZ2‐O4‐C2SAc‐ Immunogens and G6PDH Conjugates (FIG. 7)

[0205] BrAcSu (8 mg, 0.033 mmol) was dissolved in DMF (400 µL) to make a 20 mg / mL  solution.

[0206] XLZ2 Immunogen syntheses – protein activation:

[0207] BrAcOVA protein activation (24a): In a vial equipped with a magnetic stir bar and  a cap, OVA (15 mg) was suspended in buffer 2 (2 mL), the protein solution was then placed in  a cold room and BrAcSu (1.65 mg 74 µL, 20x mol excess) was added dropwise. The resulting  reaction mixture was stirred for 90 mins, then it was dialyzed using buffer 8 (2x 250 mL) in a  cold room. The next day the protein was transferred into a vial equipped with a stirring bar  and a cap.

[0208] Preparation of XLZ2‐O4‐C2SAc‐OVA (25a): The activated hapten 23 (2.47 mg, 110  µL, 20x excess) was added dropwise to the protein solution. The resulting reaction mixture  was blanketed with Argon, stirred on an ice bath, and allowed to warm up to rt. and stirred  for 16‐20h. The conjugate was then purified on a Sephadex G25‐M column with buffer 4; a  fraction of 15 mL conjugate was collected which was concentrated to ~6.5 mL on an Amicon  stir cell (2.42 mg / ml). The conjugate was then filtered off through CA filters (0.20 µm).

[0209] BrAcBSA protein activation (24b): In a vial equipped with a magnetic stir bar and  a cap, BSA (20 mg) was suspended in buffer 2 (3 mL), the protein solution was then placed in  a cold room and BrAcSu (2.81 mg 125 µL, 40x mol excess) was added dropwise. The resulting  reaction mixture was stirred in a cold room for 90 mins, then it was dialyzed with buffer 8 (2x  250 mL) in a cold room. The next day the protein was transferred into a vial equipped with a  stirring bar and a cap.

[0210] Preparation of XLZ2‐O4‐C2SAc‐BSA (25b): The activated hapten 23 (3.66 mg, 163  µL, 40x excess) was added dropwise to the protein solution. The resulting reaction mixture  was blanketed with Argon, stirred on an ice bath, and allowed to warm up to rt. and stirred  for 16‐20h. The conjugate was then purified on a Sephadex G25‐M column with buffer 4, a  volume of 15 mL of conjugate was collected which was concentrated to ~8.0 mL on an Amicon  stir cell (2.60 mg / mL). The conjugate was then filtered off through CA filters (0.20 µm).

[0211] BrAcKLH protein activation (24c): In a vial equipped with a magnetic stir bar and a cap, KLH (20 mg) was suspended in buffer 2 (3 mL), the protein solution was then placed in a cold room and BrAcSu (2.23 mg 100 pL) was added dropwise. The resulting reaction mixture was stirred for 90 mins, then it was dialyzed using buffer 8 (2x 250 mL) in a cold room. The next day, the protein was transferred into a vial equipped with a stirring bar and a cap.

[0212] Preparation of XLZ2-O4-C2SAc-KLH (25c): The activated hapten 23 (100 pL) was added dropwise to the protein solution. The resulting reaction mixture was blanketed with Argon, stirred on an ice bath, and allowed to warm up to rt. and stirred for 16-20h. The conjugate was then purified on a Sephadex G25-M column with buffer 4, a fraction of 15 mL immunogen solution was collected which was concentrated to ~5.5 mL on an Amicon stir cell (cone, of 3.60 mg / mL). The conjugate was then filtered off through CA filters (0.20 pm).

[0213] Preparation of XLZ2-O4-C2SAc-G6PDH conjugates (27a-e):

[0214] G6PDH enzyme suspension (3 mL, 30 mg) was centrifuged to 9 g for 40 mins at 4°C. The supernatant was discarded; the resulting white precipitate was dissolved in buffer 1 (3.5 mL) then dialyzed with bufferl (2 x 250 ml); 3.5 mL of enzyme with a cone of 8.33 mg / mL was recovered.

[0215] The enzyme was cooled on an ice bath, then G6PDNa (30 mg) was added, the mixture was gentle mixed until all solids dissolved; then, P-NADH (30 mg) was added, the mixture was gentle mixed until all solids dissolved.

[0216] The reaction mixture was transferred to a cold room. The BrAcSu (1.62 mg, 80 pL, 30x mol excess, of a 6 mg in 300 pL DMF solution) was added dropwise. The resulting reaction was stirred for 16h in a cold room, then it was loaded into a dialysis bag and buffer exchanged with buffer 1 (2 x 250) in a cold room. The next day, the activated BrAcG6PDH enzyme was purified by G-25 M column (CV = 55 mL); a volume of 15 mL of BrAcG6PDH enzyme (26) was collected which was concentrated to 3.3 mL on Amicon stir cell (cone. = 7.67 mg / mL). The concentration was then adjusted to 5 mg / mL using buffer 1 (1.6 mL).

[0217] Hapten activation second batch for G6PDH native conjugates:

[0218] In a vial equipped with a magnetic stir bar and cap, XLZ2-O4-C2-S2 (23) (2.0 mg) was dissolved in EtOH (150 pL), then NaOAc 25 mM pH 4.3 (100 pL) was added followed by TCEP-HCI (0.81 mg, 28 pL from a 5.8 mg in 200 pL NaOAc buffer solution). The resulting reaction mixture was blanketed with Argon and stirred at rt for lh but only 75% of the thiol reduction was completed by uPLC; an additional volume 28 pL of TCEP was then added. Thereaction mixture was blanketed with Argon then placed in a cold room and stirred overnight. The next day, the reaction was completed (6.5 mg / mL activated hapten 24 solution).

[0219] The BrAcG6PDH activated enzyme was then placed in 5 vials (4 to 5 mg BrAcG6PDH enzyme each) (Table 3); the enzyme solutions were then chilled on an ice bath. The activated hapten (14) corresponding to the mol ratio of 5x to 25x respective to the enzyme was then added dropwise to each vial. The resulting reaction mixtures were blanketed with Argon, placed in a cold room and stirred for 16h. The next day, the resulting conjugates were purified on a G25-M column (CV = 55 mL) using buffer 4; the amounts in column 6 were recovered (Table 3).TABLE 3Example 7

[0220] Synthesis of XLZ2-Boc-O4-C8Br (29) (FIG. 8): In a vial equipped with a magnetic stir bar and a cap, N,N'-l,2-Ethanediylbis[2-bromoacetamide] linker (28) (80 mg, 0.266 mmol, 1.5 eq.) was dissolved in DMF (0.4 mL), then K2CO3 (50 mg, 0.361 mmol) was added. The resulting mixture was stirred for 5 mins, then, solution of HO-4-XLZ2-Boc in DMF (60 mg, 0.178 mmol, 0.6 ml DMF) was added dropwise over 4h. The resulting reaction was stirred at 40°C for 4 days. The reaction mixture generated a mixture of ~50% mono-reacted product (the desired product) and ~20% of di reacted side-product. The volatiles were removed on a rotatory evaporator. The resulting residue was dissolved in EtOAc (20 mL) and washed with sodium phosphate monobasic saturated solution (20 mL). The organic layer was concentrated on a rotatory evaporator, to give a crude oil which was further purified by HPLC reverse phase using a C18 column. MS calcd for [C23H33BrN4O5S+1]+ 557.14, 559.14 , found 557.38, 559.39.

[0221] LC purification program: 0 - 1 min isocratic Solvent B 10%, 1- 20 min gradient Solvent B 80%, 20 - 24 min isocratic solvent B 80%, 24 - 28 min gradient Solvent B 10%. The product eluted in the interval 16 to 19 minutes, fractions 20-24 were collected, concentrated, and lyophilized to give 50 mg of product as a white powder (0.089 mmol, 50% yield).

[0222] XH NMR (600 MHz, MeOD-d4): 6.70 (s, 2H), 4.85 (s, 8H water peak), 4.44 (s, 2H), 3.84-3.82 (s, 5H), 3.44 - 3.37 (m, 4H), 3.31 (s, 5H), 2.12 (s, 6H), 1.54 (s, 9H).

[0223] Synthesis of XZ2-O4-C8Br hapten (30): In a glass vial equipped with a stir bar, XLZ2Boc-O4-C8Br (29) (50 mg, 0.089 mol) was suspended in EtOAc (300 pL). In a separate vial, acetyl bromide (80 pL, 132 mg, 1.09 mmol) was added dropwise to EtOAc / MeOH (1 / 1 v / v 300 pL) on an ice-bath. The acetyl bromide was allowed to react with MeOH for 10 mins. Then, the Boc-protected hapten-EtOAc solution (50 mg) was added dropwise. The resulting reaction mixture was stirred for 4h. The reaction mixture was concentrated on a rotatory evaporator to give 40 mg (0.088 mmol, quantitative yield) of XLZ2-O4-C8Br hapten (30) as a pale-yellow oil. MS calcd for [Ci8H25BrN4O3S+1]+ 457.09, 459.09, found 457.29 and 459.29.

[0224] Synthesis of XLZ2Boc-O4-C2NHBoc (31): HO-4XZL2-Boc (50 mg, 0.149 mmol in 0.5 mL DMF) was placed into a vial, then K2CO3 (70 mg, 0.506 mmol) was added, followed by 2- (Boc-amino) ethyl bromide linker (66.7 mg, 0.29 mmol) and KI (~20 mg). The resulting reaction mixture was blanketed with Ar, capped, and heated to 50°C for 3 days. The volatiles were removed in vacuum to give a brownish oil which was dissolved in EtOAc (20 mL) and washed with DI water (2 x 20 mL). The organic layer was then concentrated and suspended in ACN (2 mL) and purified by preparative LC to give 60 mg of XLZ2Boc-O4-C2NHBoc (0.125 mmol) in 84% yield.

[0225] Solvent A: Water +0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH

[0226] Purification LC method: 0 - 1 min isocratic Solvent B 15%, 1 - 20 min gradientSolvent B 70%, 20 min - 26 min isocratic solvent B 70%. The product eluted at 70% Solvent B (23 - 25 min). Fractions containing the product are collected, concentrated, and lyophilized overnight to give 60 mg of product (0.125 mmol, 84% yield) as a white powder.

[0227] Ms. calcd. for [C24H37N3OS+1]+ 480.25, found 480.48.

[0228] XH NMR (600 MHz, CDCI3-d): 6.58 (s, 2H), 3.98 - 3.96 (m, 2H), 3.85 (t, J = 6.84 Hz, 2H), 3.50 - 3.49 (m, 2H), 2.80 (t, J = 6.57 Hz, 2H), 2.12 (m, 8 H), 1.54 (s, 9H), 1.44 (s, 9H).

[0229] Synthesis of XLZ2-O4-C2NH2 (32): XLZ2BocO4-C2NHBoc 31 (60 mg, 0.125 mmol) was dissolved in DCM / TFA (0.3 mL, 1 / 1 v / v) and stirred for 16h. The Boc deprotection wasconfirmed by uHPLC. The volatiles were removed on a rotatory evaporator to give 55 mg of XLZ2-O-C2NH2 as TFA salt (0.108 mmol, 86% yield). This intermediate was used in the next step without any further purification. MS calcd for [Ci4H2zN3OS+1]+ 280.15, found 280.29 (ret. time 0.510 min)

[0230] Synthesis of XLZ2-O4-C6Br hapten (33): XLZ2-O-C2NH2*TFA (55 mg, 0.108 mmol) was dissolved in DMF (0.8 mL) then NEt3 (45 pL, 3 eq.) was added. The reaction's pH was measured was ~8 using pH paper. The resulting reaction mixture was cooled on an ice bath, then BrAcSu (30 mg, 0.127 mmol, 250 pL of a 120 mg / mL BrAcSu-DMF solution) was added dropwise over 5 minutes, followed by additional NEts (20 pL). The reaction mixture was allowed to warm up to rt.; after 10 minutes the product formation was confirmed by uPLC. MS calcd for [CieH22BrN3O2S+1]+ 401.34+ ACN (41) , found 442.27 as Acetonitrile adduct, ret. time (2.37 min.)

[0231] The product was purified by preparative LC. The product eluted as a broad peak, fractions containing the product were pulled out, concentrated, and lyophilized overnight, to give 10 mg of product (0.025 mmol) in 20% yield.

[0232] Solvent A: Water + 0.1% TFA, Solvent B Acetonitrile

[0233] Purification LC program: 0 - 2 min isocratic Solvent B 10%, 2 - 20 min gradient Solvent B 60%, 20 - 23 min isocratic Solvent B 60%, 23 - T1 min gradient Solvent B 10%, 27 - 28 min isocratic solvent B 10%. The product eluted 7 to 14 mins as a broad peak.

[0234] XH NMR (600 MHz CD3CN-C / 3): 6.82 (s, 2H), 4.59 (t, s, 2H), 4.07 (t, 1 = 5.42 Hz, 2H), 3.84 - 3.82 (m, 4H), 3.58 - 3.56 (m, 2H), 3.33 - 3.31 (m, 2H), 2.57 (s, 3H), 2.21 (s, 6H). Isolated as Acetate salt.Example s: Preparation of XLZ2-O4-C8-3K G6PDH (34) and XLZ2-O4-C6-3K G6PDH (35) (FIG. 9):

[0235] The 3K G6PDH enzyme (1 mL, 10.8 mg / mL) was loaded into a dialysis bag and buffer exchanged with buffer 6 (2 x 250 mL, 4h in between the exchanges) in a cold room. The concentration was then adjusted to 5 mg / mL using buffer 6. The final volume of the enzyme solution was 2 mL.

[0236] The enzyme reduction: In a plastic tube, the 3K G6PDH enzyme solution (2 mL) was placed then DTT (20 pL, 0.5M solution) was added. The resulting reaction mixture was blanketed with Ar and rocked in a cold room for 16h. The next day, buffer 7 was degassed to30 min in a cold room, then the enzyme was dialyzed with buffer 7 (250 mL) in a cold room, then buffer exchanged using a Sephadex G25M column (CV=28 mL) with buffer 7. After column, a volume of 7 mL enzyme was recovered which was then concentrated to 2.6 mL (c =5.13 mg / mL) on an Amicon Stir cell (MW CO 30,000 Da). The enzyme solution was then placed into two vials (4 mg enzyme each), blanketed with Ar, and chilled on an ice bath.

[0237] The haptens were then dissolved in DMF to make 10 mg / mL solutions. For this, the XLZ2-O4-C6-Br hapten (1.22 mg) was dissolved in DMF (0.122 mL) and the XLZ2-O4-C8-Br hapten (1.40 mg) was dissolved in DMF (0.140 mL).

[0238] Each hapten-DMF solution was then added dropwise to the corresponding reduced enzyme (4 mg each) at 40x mol excess (column 2, Table 4); the resulting reaction mixtures were blanketed with Ar and stirred overnight in a cold room. The conjugates were then purified by Sephadex G25M column (CV 40 mL) using buffer 4, filtered through Corning CA filters 0.20 pm, and concentrated to the volumes indicated in Column 3, Table 4.TABLE 4Example 9

[0239] FIGS. 10-11 contain further schemes for synthesis of certain non-limiting embodiments of xylazine haptens / immunogens / conjugates in accordance with the present disclosure. Non-limiting examples of "Proteins" that may be conjugated thereto include KLH, BSA, OVA, G6PDH, and any carrier and / or label proteins.

[0240] Preparation of XLZ3-CH2-NH2 (39) (FIG. 12): Synthesis of XLZ3-CH2-NH2 (39): 1,1- DimethylethylN-[3-amino-2-(hydroxymethyl)propyl] carbamate (37) (125 mg, 0.61 mmol) was dissolved THF (620 pL). The solution was cooled on an ice bath, then 2,6-dimethylphenyl isothiocyanate (38) (100 pL, 101 mg, 0.98 eq. 0.6 mmol) was added dropwise over 5 minutes. The resulting reaction mixture was allowed to warm up to room temperature and stirred for 3h. The volatiles were removed on a rotatory evaporator, then DI water (200 pL), followed by HCI (10%, 600 pL), were added. The resulting reaction mixture was heated under reflux for2h. The formation of XLZ3‐CH2NH2 (39) was confirmed by uPLC. Mz /  calcd for [C13H20N3S+] 250.14,  found 250.13.   NaOH  (5N) was then added dropwise until the pH ~ 10, then Ethyl Acetate  (2x20 mL) was  used  to  extract  intermediate  39.  The  solvent was  removed  on  a rotatory evaporator to give 160 mg (0.64 mmol) of crude 39. Compound 39 was too polar to  be purified by either normal or reverse phase.

[0241] Compound 39 (160 mg, 0.64 mmol) was suspended in DMF (500 µL), then Boc2O  (640 µL, 2 eq., 1.28 mmol, 2M solution in THF) and NEt3 (93 µL, 0.66 mmol) were added. The  resulting reaction mixture was stirred for 4h to form the Boc‐XLZ3‐CH2‐NHBoc (40). m / z calcd for  [C23H36N3O4S+Na] 472.24,  found 472.31.  Intermediate 40 was then purified by LC using Ethyl Acetate / Hexanes to give 201 mg (0.45 mmol) of the Boc‐XLZ3‐CH2‐NHBoc as colorless  oil in 74% yield.

[0242] 1H NMR (600 MHz, CDCl3‐d) 6.95 (d, J = 7.01 Hz, 2H), 6.86 (t, J = 7.48 Hz, 1H), 4.96  (s, 1H), 3.74 – 3.66 (m, 2H), 3.16 – 3.06 (m, 2H), 2.76 – 2.72 (m, 1H), 2.56 – 2.52 (m, 1H), 2.44  – 2.42 (m, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 1.48 (s, 9H), 1.45 (s, 9H).

[0243] Boc‐XLZ3‐CH2‐NHBoc (40) (201 mg, 0.45 mmol) was then suspended in DCM / TFA  1 / 1 v / v 1 mL and stirred overnight. The volatiles were removed in vacuum to give 39 TFA (120  mg, 0.33 mmol) as a pale‐yellow oil (as TFA salt).

[0244] Synthesis of XLZ3‐C7‐S2 hapten (42) Formula XXXV (Fig 12): Compound 39 TFA  (35 mg, 0.096 mmol) was dissolved DMF (500 µL), NEt3 (41 µL, 3 eq.) was added followed by  4‐butyrothiolactone (41) (15 µL, 1.5 eq.). The resulting reaction mixture was heated at 60°C  for 3 days. The volatiles were removed in vacuum, then EtOH (200 µL) was added followed by I2  (catalytical). The resulting reaction mixture was heated  to 40°C  for 3h,  then purified by reverse phase LC  to give XLZ3‐C7‐S2  (42) 13 mg  in 43% yield white crystals. M / z calcd  for [C34H49N6O2S4+], 701.28, found [m+2 / 2] = 351.49.

[0245] Solvent A: Water +0.1 AcOH, Solvent B: ACN + 0.1% AcOH, UV detector at 237 nm.

[0246] LC purification program: 0 – 2 min  isocratic Solvent B 10%; 2 – 20 min gradient Solvent B 70%, 20 – 24 min gradient Solvent B 70%, 24 – 28 min isocratic Solvent B 10%.

[0247] 1H NMR (600 MHz, CD3CN‐d3) 8.40 (s, 1H), 7.11 – 7.68 (m, 6H), 6.80 (s, 2H), 3.46  (dd, J = 13.35, 2.9 Hz, 2H), 3.26 (t, J = 6.41 Hz, 4H), 3.18 (dd, J = 13.3, 8.4 Hz, 2H), 3.05‐3.02  (m, 2H), 2.88 (dd, J = 12.41, 8.82 Hz, 2H), 2.74 (t, J = 7.3 Hz, 4H), 2.28 – 2.26 (m, 6H), 2.19‐2.18  (m, 12H), 1.95 ‐1.93 (m, 6H).

[0248] Bioconjugations of XLZ3-C7-S2 hapten (42, Formula XXXV) have been conducted as described in a previous similar procedure (FIG. 7) to give conjugates with Formula XXXVI as described in FIG. 12.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0249] Illustrative embodiment 1. A composition, comprising a compound having the structure of Formula I:Formula I with the proviso that at least one of Ri, R2, and R3 is not H.

[0250] Illustrative embodiment 2.The composition of Illustrative embodiment 1, wherein the at least one of Ri, R2, and R3 that is not H comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non-bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyloxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

[0251] Illustrative embodiment 3. The composition of Illustrative embodiment 1 or 2, wherein the compound comprises the structure of one of Formulas II, III, IV, V, XI, XII, XIII, XIV,ormu aXV, XVI, XVII, XVIII, XIX, XXII, or XXIX:Formula XIII Formula XIV Formula XVFormula XVIII Formula XIX Formula XXIIFormula XXIX

[0252] Illustrative embodiment 4. The composition of any of Illustrative embodiments 1- 3, wherein the compound has the structure of one of Formulas VI, VII, VIII, IX, XX, XXI, XXIII, or XXX:Formula VI Formula VIIFormula XX Formula XXI Formula XXIHFormula XXX wherein R4 is a peptide or protein.

[0253] Illustrative embodiment 5. The composition of Illustrative embodiment 4, wherein R4 is an immunogenic carrier.

[0254] Illustrative embodiment 6. The composition of Illustrative embodiment 5, wherein the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, a polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), a carrier protein, a label protein, and combinations thereof.

[0255] Illustrative embodiment 7. A composition, comprising a compound having the structure of Formula X:wherein one of Rs and Re is present and comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non-bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyl oxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

[0256] Illustrative embodiment 8. The composition of Illustrative embodiment 7, wherein the compound has the structure of one of Formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI:Formula XXVIHFormula XXXII Formula XXXIVwherein R4 is a peptide or protein.

[0257] Illustrative embodiment 9. The composition of Illustrative embodiment 7 or 8, wherein Rs or Re comprises an immunogenic carrier.

[0258] Illustrative embodiment 10. The composition of Illustrative embodiment 9, wherein the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, a polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), a carrier protein, a label protein, and combinations thereof.

[0259] Illustrative embodiment 11. The composition of any one of Illustrative embodiments 1-10, further comprising at least one excipient.

[0260] Illustrative embodiment 12. A method of preparing an antibody, comprising the steps of: immunizing an antibody-producing animal with an immunogen comprising the composition of any one of Illustrative embodiments 1-11; and isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen.

[0261] Illustrative embodiment 13. The method of Illustrative embodiment 12, wherein the step of isolating antibodies further comprises the steps of: isolating antibody-producing spleen cells from the antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to the immunogen; and preparing a hybrid cell line by fusing the selected antibody-producing spleen cell(s) from the antibody-producing animal with an appropriate fusion partner, whereby the hybrid cell line secretes the monoclonal antibody.

[0262] Illustrative embodiment 14. A conjugate composition, comprising: the composition of any one of Illustrative embodiments 1-11; and an enzyme coupled to the composition.

[0263] Illustrative embodiment 15. The conjugate composition of Illustrative embodiment 14, wherein the enzyme is selected from the group consisting of glucose-6- phosphate dehydrogenase (G6PDH), alkaline phosphatase, p-galactosidase, and horseradish peroxidase.

[0264] Illustrative embodiment 16. The conjugate composition of Illustrative embodiment 15, wherein the enzyme is G6PDH.

[0265] Illustrative embodiment 17. An antibody produced by the method of Illustrative embodiment 12 or 13.

[0266] Illustrative embodiment 18. A kit, comprising the antibody of Illustrative embodiment 17.

[0267] Illustrative embodiment 19. The kit of Illustrative embodiment 18, further comprising the conjugate composition of any of Illustrative embodiments 14-16.

[0268] Illustrative embodiment 20. A method of detecting xylazine in a sample, the method comprising the steps of: combining a sample with the antibody of Illustrative embodiment 17; and detecting the presence of xylazine in the sample based on the specific binding of the antibody thereto.

[0269] Illustrative embodiment 21. A method of detecting xylazine in a sample, the method comprising the steps of: combining a sample with the Illustrative embodiment ofclaim 19; detecting the presence of an enzymatic reaction product of the conjugate; and determining that xylazine is present in the sample when a decrease in the enzymatic reaction product of the conjugate is observed when compared to enzymatic reaction product detected in the absence of sample.

[0270] Thus, in accordance with the present disclosure, there have been provided compositions, kits, devices, and systems, 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.

Claims

CLAIMS1. A composition, comprising a compound having the structure of Formula I:Formula I with the proviso that at least one of Ri, R2, and R3 is not H.

2. The composition of claim 1, wherein the at least one of Ri, R2, and R3 that is not H comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non-bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyloxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

3. The composition of claim 1, wherein the compound comprises the structure of one ofFormulas II, III, IV, V, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XXII, or XXIX:ormu aFormula XIH Formula XIV Formula XVFormula XVIII Formula XIX Formula XXIIFormula XXIX4. The composition of claim 1, wherein the compound has the structure of one ofFormulas VI, VII, VIII, IX, XX, XXI, XXIII, or XXX:Formula XXX wherein R4 is a peptide or protein.

5. The composition of claim 4, wherein R4 is an immunogenic carrier.

6. The composition of claim 5, wherein the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, apolysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), a carrier protein, a label protein, and combinations thereof.

7. A composition, comprising a compound having the structure of Formula X:wherein one of Rs and Re is present and comprises at least one of an aliphatic, a hydrocarbyl, a bulky organic, a non-bulky organic, an alkyl, an alkenyl, an alkynyl, a phenyl, an aryl, a substituted aryl, a carbocycle, a heterocycle, a hydrocarboxyloxy, a lower alkoxy, an aryl hydrocarbyl, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or combinations thereof.

8. The composition of claim 7, wherein the compound has the structure of one ofFormulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI:Formula XXVFormula XXVIIIFormula XXXII Formula XXXIVwherein R4 is a peptide or protein.

9. The composition of claim 7 , wherein R5 or Re comprises an immunogenic carrier.

10. The composition of claim 9, wherein the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, a polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), a carrier protein, a label protein, and combinations thereof.

11. The composition of any one of claims 1-10, further comprising at least one excipient.

12. A method of preparing an antibody, comprising the steps of: immunizing an antibody-producing animal with an immunogen comprising the composition of any one of claims 1-11; isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen.

13. The method of claim 12, wherein the step of isolating antibodies further comprises the steps of: isolating antibody-producing spleen cells from the antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to the immunogen; preparing a hybrid cell line by fusing the selected antibody-producing spleen cell(s) from the antibody-producing animal with an appropriate fusion partner, whereby the hybrid cell line secretes the monoclonal antibody.

14. A conjugate composition, comprising: the composition of any one of claims 1-11; and an enzyme coupled to the composition.

15. The conjugate composition of claim 14, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase, p- galactosidase, and horseradish peroxidase.

16. The conjugate composition of claim 15, wherein the enzyme is G6PDH.

17. An antibody produced by the method of claim 12 or 13.

18. A kit, comprising the antibody of claim 17.

19. The kit of claim 18, further comprising the conjugate composition of any of claims 14-16.

20. A method of detecting xylazine in a sample, the method comprising the steps of: combining a sample with the antibody of claim 17; and detecting the presence of xylazine in the sample based on the specific binding of the antibody thereto.

21. A method of detecting xylazine in a sample, the method comprising the steps of: combining a sample with the kit of claim 19; detecting the presence of an enzymatic reaction product of the conjugate; and determining that xylazine is present in the sample when a decrease in the enzymatic reaction product of the conjugate is observed when compared to enzymatic reaction product detected in the absence of sample.

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