Anti-naloxone and anti-naltrexone monoclonal antibodies and methods of production and use thereof

Abstract

Disclosed are antibodies that specifically bind to naloxone or naltrexone. Conjugates for producing the antibodies, and methods of producing the antibodies, are also disclosed. Also disclosed are methods of using the antibodies in direct assays of naloxone or naltrexone. Further disclosed are methods of using the antibodies to reduce naloxone or naltrexone interference in opiate assays.

Description

[0001] This application is a divisional application of the invention patent application with the application date of January 30, 2020, application number 202080011933.4, and invention name “Anti-naloxone and anti-naltrexone monoclonal antibodies and methods for their production and use”. Technical Field

[0002] The field of monoclonal antibodies of the present invention relates specifically to monoclonal antibodies that specifically bind to naloxone and / or naltrexone, and homogeneous immunoassays that can be used to detect the presence of these drugs in biological samples and / or provide qualitative screening and / or quantitative clinical measurement thereof. Background Art

[0003] Naloxone is commonly used as an antagonist for abused opioids, usually in cases of drug overdose. Naloxone is a short-acting, life-saving drug that relieves patients from opioid overdose by stripping opioids from opioid receptors. Repeated injections of naloxone (NARCAN® (naloxone hydrochloride), ADAPT Pharma, Inc., Radnor, PA.) are given to patients to saturate drug receptors, minimizing the pharmacological response to abused opioids. Naloxone works within minutes and lasts for about an hour.

[0004] Naltrexone is another commonly used opioid antagonist drug that is also used in alcohol abuse situations; naltrexone is an opiate / alcohol blocker that has been used for the last 30 years. Naltrexone is slow acting and lasts longer than naloxone. VIVITROL® (Alkermes, Inc., Dublin, Ireland) is an extended release form of naltrexone. The extended release form of VIVITROL® helps with patient compliance because it does not need to be used every day.

[0005] Both naloxone and naltrexone will also blunt the effects of alcohol, and therefore, they are often used after discharge from an inpatient treatment program to prevent alcohol relapse.

[0006] Naloxone is rapidly distributed throughout the body. The mean serum half-life has been shown to range from 30 to 81 minutes, which is shorter than the mean half-life of some opiates and requires repeated dosing if opioid receptors must be stopped from being triggered for a prolonged period of time. Naloxone is primarily metabolized by the liver; its major metabolite is naloxone-3-glucuronide, which is excreted in the urine.

[0007] Naltrexone is metabolized primarily in the liver by dihydrodiol dehydrogenase to 6β-naltrexol. Other metabolites include 2-hydroxy-3-methoxy-6β-naltrexol and 2-hydroxy-3-methoxy-naltrexone. These intermediates are then further metabolized by conjugation with glucuronides. The plasma half-lives of naltrexone and its metabolite 6β-naltrexol are approximately four (4) and thirteen (13) hours, respectively.

[0008] Randox Toxicology (Crumlin, United Kingdom) provides a naloxone assay based on the BioChipArrayTechnology (BAT), which is based on the enzyme-linked immunosorbent assay (ELISA) principle. BiochipArray Technology is a sophisticated multiplex testing platform that allows for simultaneous quantitative or qualitative detection of a wide range of analytes from a single sample. The Biochip is a solid-state device with discrete test sites to which antibodies specific for different drug compounds are fixed and stabilized. A competitive chemiluminescent immunoassay is then used, providing a highly sensitive screen. However, for this assay, the level of naltrexone cross-reactivity was 12.5%, and the level of naloxone 3-BD glucuronide cross-reactivity was 70.6%.

[0009] Immunalysis Corporation (Pomona, CA) offers an ELISA naltrexone assay for oral fluid and forensic use that is also based on the ELISA principle (see, e.g., Catalog Nos. 239-0096 and 239-0480).

[0010] Neogen Corporation (Lansing, MI) offers a Naltrexone / Nalbuphine ELISA kit, which is a qualitative one-step kit designed for use as a screening device for the detection of naltrexone, nalbuphine, and / or other metabolites. However, this test has only 4% cross-reactivity to naloxone (see, e.g., Catalog Nos. 133015 and 133019).

[0011] Other methods for detecting naloxone and naltrexone involve high performance liquid chromatography / tandem mass spectrometry (LC-MS / MS) analysis. For example, NMS Labs (Willow Grove, PA) offers LC-MS / MS analysis for naltrexone (total) and the metabolite 6-β-naltrexol (total) in urine (see, e.g., Test Code 3116U, Test Name Naltrexone and Metabolites – Total (Conjugated / Unconjugated), Urine).

[0012] However, there are currently no known homogeneous immunoassays that can be used for one or both of these two drugs. Therefore, there is a need in the art for monoclonal antibodies that specifically bind to naloxone and / or naltrexone, and homogeneous immunoassays that can be used to detect the presence of these drugs in biological samples and / or provide qualitative screening and / or quantitative clinical measurement thereof.

[0013] Furthermore, given the structural similarities between naloxone and naltrexone and various opiates (see Figure 1 ), which drugs can cross-react in various opiate assays. Specifically, it is often observed that various opiate assays provide false positive results for patients undergoing naloxone / naltrexone treatment. Therefore, there is a need in the art to neutralize naloxone and naltrexone present in biological samples to thereby prevent any false positive or falsely elevated opiate measurements, thereby providing more accurate qualitative screening and quantitative measurements in clinical opiate assays. Summary of the invention

[0014] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it should be understood that the present disclosure is not limited to the application of the details of the construction and arrangement of the components set forth in the following description. The present disclosure can have other embodiments, or can be practiced or implemented in various ways. Therefore, 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. Moreover, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0015] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. Typically, the terms and techniques associated with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reaction and purification techniques are performed according to the manufacturer's instructions or as usually completed in the art or as described herein. The aforementioned techniques and procedures are typically performed according to conventional methods well-known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Coligan et al. Current Protocols in Immunology (Current Protocols, Wiley Interscience (1994)), which are incorporated herein by reference. Nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0016] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the technical levels of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein 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.

[0017] In view of the present disclosure, all compositions, kits and / or methods disclosed herein can be prepared and performed without undue experimentation. Although the compositions, kits and / or methods have been described in terms of specific embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions, kits and / or methods, and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined in the appended claims.

[0018] As utilized in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings: When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Thus, the terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" may refer to one or more compounds, 2 or more compounds, 3 or more compounds, 4 or more compounds, or a greater number of compounds. The term "multiple" means "two or more".

[0019] The use of the term "at least one" will be understood to include one and any number more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to as many as 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 is not considered restrictive, because higher limits can 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, and any combination of X, Y and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is only for the purpose of distinguishing two or more items, and is not meant to imply, for example, any order or sequence or importance of one item relative to another, or any order of addition.

[0020] The term "or" used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0021] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a specific element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "an example" appearing in various places in the specification do not necessarily all refer to the same embodiment. In addition, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the composition / instrument / device, method used to determine the value, or the variation that exists between study subjects. For example, but not limited to, when the term "about" is utilized, the specified value may vary from the indicated value by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, as such variations are applicable to performing the disclosed methods and are understood by those of ordinary skill in the art.

[0023] As used in the specification and claims, 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.

[0024] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before 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 also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing with this example, expressly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. The skilled artisan will understand that, unless apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0025] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely or that the subsequently described event or circumstance occurs to a large extent or degree. For example, when related to a specific event or environment, 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" can mean that two items are 100% adjacent to each other, or that two items are in close proximity to each other, but are not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item, but is in close proximity to the other item.

[0026] The terms "analog" and "derivative" are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functionality in their structure as a given compound, but may also contain one or more substitutions thereto. As used herein, the term "substitution" 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 iodite, a C1-C4 compound selected from one of the following: an optionally substituted straight, branched or cyclic alkyl, and a straight, branched or cyclic alkenyl, wherein the optional substituent is selected from one or more alkenyl alkyl, alkynyl alkyl, cycloalkyl, cycloalkenyl alkyl, aryl alkyl, heteroaryl alkyl, heterocycloalkyl, optionally substituted heterocycloalkenyl alkyl, aryl cycloalkyl and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituents are selected from one or more of the following: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxy, and -C(O))-alkyl.

[0027] The term "sample" as used herein will be understood to include any type of biological sample that can be utilized according to the present disclosure. Examples of fluid biological samples that can 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, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0028] The term "specific binding partner" as used herein is understood to refer to any molecule that can specifically associate with a macrophilin-binding drug for the purpose of its detection. For example, but not by way of limitation, the specific binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix) or any combination and / or derivative thereof, as well as any other molecule that can specifically bind to a macrophilin-binding drug.

[0029] The term "antibody" is used in the broadest sense herein and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments thereof and conjugates thereof that exhibit the desired analyte binding biological activity (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of the intact antibody), antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. The antibody may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA1, and IgA2).

[0030] The terms "peptide", "polypeptide" and "protein" are used herein to refer to a polymer of amino acid residues. The term "polypeptide" as used herein is a general term referring to a native protein, protein fragment or analog of a polypeptide sequence. Therefore, native proteins, protein fragments and analogs are species of the genus polypeptide. The term "isolated peptide / polypeptide / protein" as used herein refers to a peptide / polypeptide / protein of cDNA, recombinant RNA or synthetic origin or some combination thereof, which, depending on the nature of its origin or the source from which it is derived: (1) is not associated with peptides / polypeptides / proteins found in nature, (2) is free of other peptides / polypeptides / proteins from the same source, for example, does not contain mouse proteins, (3) is expressed by cells from a different species, and / or (4) does not exist in nature.

[0031] As used herein, the term "amino acid" includes all molecules, whether natural or synthetic, that include both an amino functional group and an acid functional group and that are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0032] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotides may be further modified, such as by conjugating with a labeling component. The terms "isolated nucleic acid" and "isolated polynucleotide" are used interchangeably; a nucleic acid or polynucleotide is considered "isolated" if it: (1) is not associated with all or part of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0033] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0034] The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polynucleotide or polypeptide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory or otherwise is naturally occurring. The term "naturally occurring" may be used interchangeably herein with the term "native".

[0035] The term "selective hybridization" as referred to herein means detectably and specifically combined. Polynucleotides, oligonucleotides and fragments thereof encoding peptides / polypeptides / proteins according to the inventive conception are selectively hybridized with nucleic acid chains under conditions of hybridization and washing that minimize the estimable amount of detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides and fragments of the inventive conception and the target nucleic acid sequence will be at least 80%, and most commonly have at least 85%, 90%, 95%, 99% and 100% increased homology. If there is partial or complete identity between two amino acid sequences, they are homologous. For example, when two sequences are compared for maximum matching, 85% homology means that 85% of the amino acids are the same. Allowing spaces (either of the two sequences of the match) in maximum matching; 5 or less space lengths are preferred (but not limited), and 2 or less are more preferred (but not limited). Alternatively, as the term is used herein, two protein sequences (or polypeptide sequences derived therefrom of at least 30 amino acids in length) are homologous if they have an alignment score greater than 5 (in standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. See Dayhoff, MO, Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. When optimally aligned using the ALIGN program, two sequences are more preferably homologous if the amino acids of the two sequences or portions thereof are greater than or equal to 50% identical. The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous to all or part of a reference polynucleotide sequence (i.e., identical, not strictly evolutionarily related), or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that a complementary sequence is homologous to all or part of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0036] The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is defined as a sequence used as a basis for sequence comparison; a reference sequence can be a subset of a larger sequence, for example, a segment of a full-length cDNA or gene sequence given in a sequence listing or can comprise the entire cDNA or gene sequence. Typically, a reference sequence is at least 18 nucleotides or 6 amino acids long, often at least 24 nucleotides or 8 amino acids long, and often at least 48 nucleotides or 16 amino acids long. Because two polynucleotide or amino acid sequences can each (1) contain a sequence that is similar between the two molecules (i.e., a portion of the complete polynucleotide or amino acid sequence), and (2) can further contain a sequence that is different between the two polynucleotide or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by aligning the sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a conceptual segment of at least 18 consecutive nucleotide positions or 6 amino acids, wherein a polynucleotide sequence or an amino acid sequence can be compared to a reference sequence of at least 18 consecutive nucleotides or 6 amino acid sequences, and wherein the portion of the polynucleotide sequence in the comparison window may contain 20% or less additions, deletions, substitutions, etc. (i.e., gaps) when compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences over the comparison window can be performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443 (1970)), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), or by inspection, and the best alignment (i.e., that resulting in the highest percentage of homology over the comparison window) produced by each method is selected.

[0037] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical over a comparison window (i.e., on a nucleotide-to-nucleotide or residue-to-residue basis). The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in the two sequences to produce a matching number of positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce a sequence identity percentage. As used herein, the term "substantial identity" refers to a characteristic of a polynucleotide or amino acid sequence wherein the polynucleotide or amino acid comprises a sequence having at least 85% sequence identity, such as at least 90% to 95% sequence identity, or at least 99% sequence identity compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, often a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the sequence identity percentage is calculated by comparing the reference sequence over the comparison window to a total of 20% or less of the reference sequence that may include deletions or additions. The reference sequence may be a subset of a larger sequence.

[0038] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. 。 See Immunology--A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. 20 conventional amino acids, non-natural amino acids such as: α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acid stereoisomers (e.g., D-amino acids) can also be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In polypeptide notation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0039] As applied to polypeptides, the term "substantial identity" means that two peptide sequences share at least 80% sequence identity, such as at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity when optimally aligned (such as by the program GAP or BESTFIT using default gap weights). In certain specific (but non-limiting) embodiments, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxy side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Specific conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

[0040] The term "variant" of a reference polypeptide refers to a polypeptide having one or more amino acid substitutions, deletions or insertions relative to a reference polypeptide. Amino acid substitutions may be "conservative" or "non-conservative". A "conservative" amino acid substitution refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (such as, but not limited to, size and charge). Conservative substitutions are those that occur within a family of amino acids, which are related by their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More specific families are: serine and threonine are the aliphatic-hydroxyl family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine and isoleucine are the aliphatic family; and phenylalanine, tryptophan and tyrosine are the aromatic family. For example, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, isolated substitutions of aspartic acid with glutamic acid, isolated substitutions of threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitutions do not involve amino acids within framework sites. Whether an amino acid change results in a functional peptide can be easily determined by measuring the specific activity of the polypeptide derivative. Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those of ordinary skill in the art. The amino- and carboxyl-termini of preferred fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or private sequence databases. In specific (but not limiting) embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein configuration domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al. Science, 253:164 (1991)). Thus, the above examples show that one skilled in the art can identify sequence motifs and structural configurations that can be used to define structural and functional domains according to the present disclosure.

[0041] Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify the physicochemical or functional properties of such analogs. Analogs may include various mutations of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (such as, but not limited to, conservative amino acid substitutions) may be made in the naturally occurring sequence (such as, but not limited to, in portions of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the replacing amino acid should not tend to disrupt helices present in the parent sequence, or interrupt other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure © (Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. (Nature 354:105 (1991)), each of which is incorporated herein by reference.

[0042] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence. A polypeptide fragment may be any length less than the length of a reference polypeptide.

[0043] The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit desired biological activity. Therefore, the term "antibody" or "antibody peptide" refers to a full-length immunoglobulin molecule (i.e., a complete antibody), or a binding fragment thereof that competes with a complete antibody for specific antigen binding. The binding fragment can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. The binding fragment includes Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®) and other antibody fragments that retain at least a portion of the variable region of a complete antibody. See, for example, Hudson et al. ( Nature Med. , 9:129-134(2003)).

[0044] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more antibody fragments that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[0045] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0046] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0047] The term "CDR" and its plural "CDRs" refer to the complementary determining region (CDR) of an antibody or antibody fragment, which determines the binding characteristics of the antibody or antibody fragment. In most cases, three CDRs are present in the light chain variable region (CDRL1, CDRL2 and CDRL3), and three CDRs are present in the heavy chain variable region (CDRH1, CDRH2 and CDRH3). The CDRs contribute to the functional activity of the antibody molecule and are separated by the amino acid sequence comprising the scaffold or framework region. Among the individual CDRs, the CDR3 sequence, particularly CDRH3, is the most diverse and therefore has the strongest contribution to antibody specificity. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), which is incorporated by reference in its entirety); and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature , 342:877 (1989), which is incorporated by reference in its entirety).

[0048] The term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or a T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitope determinants typically include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups. In certain embodiments, an epitope can have specific three-dimensional structural features (e.g., "conformational epitopes") and specific charge characteristics.

[0049] An epitope is defined as being "identical" to another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise the same epitope. In certain embodiments, the same epitope may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to the same epitope.

[0050] When an antibody preferentially recognizes an antigen in a complex mixture of proteins and / or macromolecules, it "specifically binds" to that antigen. In certain embodiments, an antibody comprises an antigen binding site that specifically binds to a particular epitope. In certain such embodiments, an antibody is capable of binding to different antigens, as long as the different antigens contain that particular epitope or a closely related epitope. In certain cases, for example, homologous proteins from different species may contain the same epitope. In certain embodiments, an antibody is present in an amount of no more than 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 The antibody specifically binds to the antigen with a dissociation constant of M. When the antibody specifically binds to the receptor or ligand (i.e., counter-receptor), it can substantially inhibit the adhesion of the receptor to the ligand. As used herein, the antibody substantially inhibits the adhesion of the receptor to the ligand (as measured in an in vitro competitive binding assay) when an excess of the antibody reduces the amount of receptor bound to the ligand by at least about 20%, 40%, 60%, or 80%, 85%, or 90%.

[0051] "Separated" antibodies are antibodies that have been separated and / or recovered from the components of the environment in which they are produced. The contaminating components of the environment in which they are produced are materials that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-protein solutes. In certain embodiments, as measured by at least three different methods, the antibody will be purified to: 1) as determined by the Lowry method, by the weight of the antibody, greater than 50% by weight, 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) sufficient to obtain at least 10 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, such as the degree of at least 15 residues of the sequence; or 3) homogeneous, by using Coomassie blue or alternatively silver-stained SDS-PAGE under reducing or non-reducing conditions. The separated antibody includes the in situ antibody in the recombinant cell, because at least one component in the environment in which the antibody is produced will not exist. However, the separated antibody will usually be prepared by at least one purification step. In addition, "separated antibodies" are substantially free of other antibodies with different antigenic specificities. However, the separated antibody can have certain cross-reactivity with other related antigens.

[0052] The term "antibody mutant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such mutants must have less than 100% sequence identity or similarity with the amino acid sequence of the heavy chain or light chain variable domain of the antibody, at least 75% amino acid sequence identity or similarity with the amino acid sequence, such as at least 80%, or at least 85%, or at least 90%, or at least 95% amino acid sequence identity or similarity.

[0053] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population that specifically binds to the same epitope, that is, each antibody constituting the population is identical except for the possible naturally occurring mutation that may exist in a small amount. Contrary to conventional (polyclonal) antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. Except for their specificity, monoclonal antibodies are advantageous because in a production method, they can be synthesized by hybridoma culture, and are therefore not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring antibodies to be produced by any ad hoc method. For example, in one embodiment, the monoclonal antibody produced according to the disclosure can be prepared by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0054] The monoclonal antibodies utilized in accordance with the present disclosure may be produced by any method known in the art, including, but not limited to, the result of an active immunization regimen; the result of an immune response that results in the natural production of antibodies during a disease or cancer; phage-derived antibodies; etc. In addition to the hybridoma production methods listed above, the monoclonal antibodies of the present disclosure may be produced by various other 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 phage display libraries (see, e.g., Clackson et al., Nature , 352:624-628 (1991); and Marks et al., J. Mol. Biol ., 222:581-597 (1991)); and various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988), Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0055] Once antibodies have been obtained, e.g., once individual B cells have been identified and / or monoclonal antibodies have been produced, sequences encoding the variable regions of these antibodies can be obtained. Variable region sequences can be obtained, for example, by first sequencing the antibody protein produced by a hybridoma, B-cell, or phage and determining the encoding nucleic acid sequence. In one embodiment, immunoglobulin variable region (VH and VL) DNA or cDNA can also be sequenced. When antibodies are derived from hybridoma cell lines or isolated B-cells, PCR can be used, for example, by Babcook et al. ( Proc. Natl. Acad. Sci. USA cDNA encoding the variable region was amplified using the methods described in WO 92 / 02551 (1996), 93:7843-7848 (1996) and PCT Publication No. WO 92 / 02551. The contents of both references are expressly incorporated herein by reference in their entirety.

[0056] The term "neutralizing antibody" or "neutralizing antibody" refers to an antibody that reduces at least one activity of a polypeptide comprising an epitope to which the antibody specifically binds. In certain embodiments, the neutralizing antibody reduces in vitro and / or in vivo activity.

[0057] The term "antigen binding site" refers to the portion of an antibody that is capable of specifically binding to an antigen. In certain embodiments, the antigen binding site is provided by one or more antibody variable regions.

[0058] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition). Typically, a substantially pure composition will contain more than about 50%, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In one embodiment, the target species is purified to substantial homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0059] The term "agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material. In certain embodiments, an "agent" may be a monoclonal antibody according to the present disclosure.

[0060] The term "antagonist" refers to an agent that decreases the activity of a protein / enzyme.

[0061] The term "agonist" refers to an agent that increases the activity of a protein / enzyme.

[0062] The term "carrier protein" as used herein should be understood to refer to an immunogenic protein that can be attached to small molecules (such as (but not limited to) drugs, organic compounds, and peptides and oligosaccharides with a molecular weight of less than 2-5 kDa) for the purpose of stimulating an immune system response in the form of antibodies produced against the small molecules, which are not generally immunogenic.

[0063] Turning now to the inventive concepts, certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone and / or naltrexone. These two compounds are depicted below as Formulas I and II, respectively.

[0064] In a specific non-limiting embodiment, the antibody or functional fragment thereof specifically binds to a conjugate comprising naloxone and / or naltrexone bound to a carrier protein. The conjugate may contain any carrier protein known in the art or otherwise contemplated herein, so long as the carrier protein: (1) can be conjugated to naloxone and / or naltrexone; (2) is immunogenic; and (3) can function to stimulate an immune system response in the form of antibodies to naloxone and / or naltrexone. Non-limiting examples of carrier proteins that can be utilized in accordance with the present disclosure include KLH (keyhole limpet hemoglobin); Hemocyanin), BSA (bovine serum albumin), ovalbumin, BTG (bovine thyroglobulin), and BGG (bovine gamma globulin).

[0065] In certain non-limiting embodiments, the carrier protein is attached to a specific carbon position on naloxone and / or naltrexone. For example (but not by way of limitation), the carrier protein can be attached to the 1-carbon, 2-carbon, 3-carbon, 6-carbon, 7-carbon, or 8-carbon position of naloxone and / or naltrexone. Non-limiting examples of these conjugate immunogens are shown in Formulas III-VIII below. Note that each of these formulas encompasses both naloxone and naltrexone, and the carrier protein is illustrated as being attached to a different carbon in each formula (Formula III: 3-carbon; Formula IV: 1-carbon; Formula V: 2-carbon; Formula VI: 6-carbon; Formula VII: 8-carbon; and Formula VIII: 7-carbon).

[0066] Wherein, in Formula III: A is a heteroatom (such as, but not limited to: -O-, -N- or -S-); B is a functional group (such as, but not limited to: -CO-, a lower alkyl group (C0 to C 10 ), -CONH-, -SO2-, -PO4-, etc.); C is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH or poly (amino acid)); and R2 is H, alkyl, allyl or methylenecyclopropyl.

[0067] Wherein, in Formula IV: D is a heteroatom (such as, but not limited to: O-, S-, CO- or CH2-); E is a lower alkyl, CO2-, CONH-, SO2- or PO4-; F is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH or poly (amino acid)); R2 is H, alkyl, allyl or methylenecyclopropyl; and R1 is H or CH3.

[0068] Wherein, in Formula V: D is a heteroatom (such as, but not limited to: O-, S-, CO- or CH2-); E is a lower alkyl, CO2-, CONH-, SO2- or PO4-; F is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH or poly (amino acid)); R2 is H, alkyl, allyl or methylenecyclopropyl; and R1 is H or CH3.

[0069] Wherein, in Formula VI: G is a linking group, which includes at least two of the following: a lower alkyl chain, CO2-, CONH-, SO2- and PO2-; H is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH or poly (amino acid)); R1 is H or CH3; and R2 is H, alkyl, allyl or methylenecyclopropyl.

[0070] Wherein, in Formula VII: I is a heteroatom (such as, but not limited to: O-, S-, CO- or CH2-); J is a lower alkyl chain, CO2-, CONH-, SO2- or PO4-; K is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH or poly (amino acid)); R1 is H or CH3; and R2 is H, alkyl, allyl or methylenecyclopropyl.

[0071] Wherein, in Formula VII: L is a heteroatom (such as, but not limited to O, S, CO or CH2); M is a lower alkyl chain, CO2-, CONH-, SO2- or PO4-; N is a carrier protein (such as, but not limited to: KLH, BSA, OVA, BGG, G6PDH, etc.); R1 is H or CH3; and R2 is H, alkyl, allyl or methylenecyclopropyl.

[0072] In a specific (but non-limiting) embodiment, the antibody or functional fragment thereof specifically binds to a conjugate comprising OVA attached to the 3-carbon position and / or the 6-carbon position of naloxone and / or naltrexone.

[0073] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is allyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 2; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 3; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 4; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 6; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 7; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 8. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0074] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 22; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 23; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 24; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 26; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 27; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 28. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0075] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 42; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 43; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 44; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 46; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 47; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 48. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0076] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 62; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 63; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 64; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 66; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 67; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 68. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0077] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 82; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 83; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 84; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 86; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 87; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 88. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0078] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 102; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 103; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 104; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 106; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 107; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 108. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0079] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 122; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 123; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 124; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 126; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 127; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 128. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0080] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 142; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 143; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 144; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 146; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 147; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 148. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0081] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 162; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 163; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 164; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 166; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 167; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 168. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0082] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 182; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 183; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 184; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 186; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 187; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 188. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0083] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 202; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 203; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 204; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 206; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 207; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 208. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0084] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 222; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 223; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 224; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 226; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 227; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 228. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0085] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone, wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 242; (ii) a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 243; (iii) a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 244; (iv) a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 246; (v) a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 247; and (vi) a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 248. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0086] In a specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain variable region, the amino acid sequence of which is at least about 70% identical to any of the heavy chain variable region amino acid sequences summarized in Tables 2-14 (i.e., SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257), such as (but not limited to) SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257. NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0087] In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain variable region, the amino acid sequence of the heavy chain variable region is the same as SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257 differs in less than about 25 amino acids, less than about 24 amino acids, less than about 23 amino acids, less than about 22 amino acids, less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, less than about 2 amino acids, or less than about 1 amino acid.

[0088] In a specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment thereof has a light chain variable region, the amino acid sequence of which is at least about 70% identical to any of the light chain variable region amino acid sequences summarized in Tables 2-14 (i.e., SEQ ID NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258), such as (but not limited to) SEQ ID NO: NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0089] In a specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a light chain variable region, the amino acid sequence of which differs from SEQ ID NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258 by less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, less than about 2 amino acids, or less than about 1 amino acid.

[0090] In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is at least about 90% identical to SEQ ID NO:17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257, and / or the antibody or its functional fragment has a light chain variable region, and the amino acid sequence of the light chain variable region is at least about 90% identical to SEQ ID NO:18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258. In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region differs from that of SEQ ID NO:17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257 by at least about 12 amino acids, and the amino acid sequence of the light chain variable region differs from that of SEQ ID NO:18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258 by at least about 12 amino acids.

[0091] In another specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237 or 257, and the amino acid sequence of the light chain variable region is SEQ ID NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238 or 258.

[0092] In another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain whose amino acid sequence is at least about 70% identical to any of the heavy chain amino acid sequences summarized in Tables 2-14 (i.e., SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241), such as (but not limited to) SEQ ID NO: NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0093] In a specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain, the amino acid sequence of which differs from SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241 by less than about 100 amino acids, less than about 90 amino acids, less than about 80 amino acids, less than about 75 amino acids, less than about 70 amino acids, less than about 65 amino acids, less than about 60 amino acids, less than about 55 amino acids, less than about 50 amino acids, less than about 45 amino acids, less than about 40 amino acids, less than about 35 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 24 amino acids, less than about 23 amino acids. , less than about 22 amino acids, less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, less than about 2 amino acids, or less than about 1 amino acid.

[0094] In a specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment thereof has a light chain, the amino acid sequence of which is at least about 70% identical to any of the light chain amino acid sequences summarized in Tables 2-14 (i.e., SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245), such as (but not limited to) SEQ ID NO: NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0095] In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a light chain, the amino acid sequence of which is the same as SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245 differs in less than about 45 amino acids, less than about 40 amino acids, less than about 35 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 24 amino acids, less than about 23 amino acids, less than about 22 amino acids, less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, less than about 2 amino acids, or less than about 1 amino acid.

[0096] In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain, the amino acid sequence of which is at least about 90% identical to SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241, and / or the antibody or its functional fragment has a light chain, the amino acid sequence of which is at least about 70% identical to SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245. In a specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain and / or a light chain, the amino acid sequence of the heavy chain differs from that of SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241 by less than about 46 amino acids, and the amino acid sequence of the light chain differs from that of SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245 by less than about 24 amino acids.

[0097] In another specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221 or 241, and the amino acid sequence of the light chain is SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225 or 245.

[0098] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment has a heavy chain encoded by a polynucleotide having at least about 70% identity to SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 (and / or a heavy chain variable region encoded by a polynucleotide having at least about 70% identity to SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259), such as, but not limited to, a polynucleotide having at least about 70% identity to SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 and / or a polynucleotide having at least about 70% identity to SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259. NO:19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity. The heavy chain and / or heavy chain variable region encoded by the polynucleotides.

[0099] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment has a light chain encoded by a polynucleotide having at least about 70% identity to SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 (and / or a light chain variable region encoded by a polynucleotide having at least about 70% identity to SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260), such as (but not limited to) a polynucleotide having at least about 70% identity to SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 and / or a light chain variable region encoded by a polynucleotide having at least about 70% identity to SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 NO:20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 having at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity encoded by polynucleotides.

[0100] In yet a further specific (but non-limiting) embodiment, the antibody or functional fragment has a heavy chain encoded by a polynucleotide having at least about 70% identity with SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249, and / or the antibody or functional fragment has a light chain encoded by a polynucleotide having at least about 70% identity with SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253.

[0101] In yet a further specific (but non-limiting) embodiment, the antibody or functional fragment has a heavy chain variable region, which is encoded by a polynucleotide having at least about 70% identity with SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259, and / or the antibody or functional fragment has a light chain variable region, which is encoded by a polynucleotide having at least about 70% identity with SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260.

[0102] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain (and / or a heavy chain variable region) encoding a sequence that differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 in that (and / or the sequence encoding the heavy chain variable region differs from SEQ ID NO: NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 differs in) less than about 100 nucleotides, less than about 90 nucleotides, less than about 80 nucleotides, less than about 75 nucleotides, less than about 70 nucleotides, less than about 60 nucleotides, less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, less than about 35 nucleotides, less than about 30 nucleotides, less than about 25 nucleotides, less than about 20 nucleotides, less than about 15 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, less than about 7 nucleotides, less than about 6 nucleotides, less than about 5 nucleotides, less than about 4 nucleotides, less than about 3 nucleotides, less than about 2 nucleotides, or less than about 1 nucleotide.

[0103] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment thereof has a light chain (and / or light chain variable region) encoding a sequence that differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 in that (and / or the sequence encoding the light chain variable region differs from SEQ ID NO: NO:20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 differs in) less than about 100 nucleotides, less than about 90 nucleotides, less than about 80 nucleotides, less than about 75 nucleotides, less than about 70 nucleotides, less than about 60 nucleotides, less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, less than about 35 nucleotides, less than about 30 nucleotides, less than about 25 nucleotides, less than about 20 nucleotides, less than about 15 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, less than about 7 nucleotides, less than about 6 nucleotides, less than about 5 nucleotides, less than about 4 nucleotides, less than about 3 nucleotides, less than about 2 nucleotides, or less than about 1 nucleotide.

[0104] In yet another specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain and / or a light chain, the sequence encoding the heavy chain differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 by less than about 100 nucleotides, and the sequence encoding the light chain differs from SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 by less than about 70 nucleotides.

[0105] In yet another specific (but non-limiting) embodiment, the antibody or its functional fragment has a heavy chain variable region and / or a light chain variable region, and the sequence encoding the heavy chain variable region differs from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 in less than about 35 nucleotides, and the sequence encoding the light chain variable region differs from SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 in less than about 34 nucleotides.

[0106] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 10; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 11; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 12; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 14; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 15; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 16. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0107] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, conjugates of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 30; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 31; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 32; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 34; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 35; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 36. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0108] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, conjugates of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 50; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 51; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 52; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 54; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 55; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 56. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0109] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, conjugates of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 70; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 71; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 72; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 74; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 75; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 76. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0110] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, conjugates of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 90; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 91; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 92; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 94; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 95; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 96. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i)-(vi).

[0111] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, a conjugate of Formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 110; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 111; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 112; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 114; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 115; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 116. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0112] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, a conjugate of Formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 130; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 131; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 132; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 134; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 135; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 136. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0113] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naltrexone (and in certain specific (but non-limiting) embodiments, a conjugate of Formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 150; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 151; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 152; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 154; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 155; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 156. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0114] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 170; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 171; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 172; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 174; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 175; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 176. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0115] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 190; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 191; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 192; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 194; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 195; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 196. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0116] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 210; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 211; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 212; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 214; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 215; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 216. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0117] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 230; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 231; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 232; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 234; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 235; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 236. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0118] Certain non-limiting embodiments of the present disclosure relate to antibodies or functional fragments thereof that specifically bind to naloxone (and in certain specific (but non-limiting) embodiments, a conjugate of formula III, wherein R2 is cyclopropyl), wherein the antibody comprises one or more of the following: (i) a heavy chain variable region CDR1 encoded by the sequence of SEQ ID NO: 250; (ii) a heavy chain variable region CDR2 encoded by the sequence of SEQ ID NO: 251; (iii) a heavy chain variable region CDR3 encoded by the sequence of SEQ ID NO: 252; (iv) a light chain variable region CDR1 encoded by the sequence of SEQ ID NO: 254; (v) a light chain variable region CDR2 encoded by the sequence of SEQ ID NO: 255; and (vi) a light chain variable region CDR3 encoded by the sequence of SEQ ID NO: 256. In a specific non-limiting embodiment, the antibody or functional fragment thereof comprises two, three, four or five of the above (i)-(vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0119] The antibody or its functional fragment may be a monoclonal antibody or its functional fragment. Alternatively, the antibody or its functional fragment may be a polyclonal antibody or its functional fragment.

[0120] In certain non-limiting embodiments, the antibody or functional fragment thereof is further defined as being selected from the group consisting of a full-length immunoglobulin molecule, scFv, Fab fragment, Fab' fragment, F(ab')2, Fv, disulfide-linked Fv and combinations thereof.

[0121] In certain non-limiting embodiments, the antibody or its functional fragment is isolated. In specific (but non-limiting) embodiments, the antibody or its functional fragment is purified.

[0122] The present disclosure also relates to antibodies or functional fragments thereof, which bind to the same epitope as any of the antibodies or functional fragments described above.

[0123] Certain non-limiting embodiments of the present disclosure also relate to methods for producing antibodies or functional fragments thereof that can specifically bind to naloxone and / or naltrexone. The methods include immunizing a non-human animal with an antigenic compound comprising a conjugate of a carrier protein attached to naloxone and / or naltrexone; and recovering the antibodies or functional fragments thereof from the plasma of the non-human animal.

[0124] Certain non-limiting embodiments of the present disclosure also relate to methods for producing antibodies or functional fragments thereof that can specifically bind to naloxone and / or naltrexone. The method comprises immunizing a non-human animal with an antigenic compound comprising a conjugate of a carrier protein attached to naloxone and / or naltrexone, thereby inducing B-cells to produce antibodies that bind to the conjugate; and obtaining antibodies or functional fragments thereof produced by the B-cells. The antibodies or functional fragments thereof can be obtained by various means known in the art, such as (but not limited to) via hybridoma technology or by B-cell PCR technology. The method may further include an optional step of further selecting antibodies or functional fragments thereof based on binding to naloxone and / or naltrexone.

[0125] Certain non-limiting embodiments of the present disclosure relate to a hybridoma producing any of the antibodies or functional fragments thereof described above.

[0126] Certain non-limiting embodiments of the present disclosure relate to methods for producing antibodies or functional fragments thereof that can specifically bind to naloxone and / or naltrexone. In the method, a hybridoma capable of producing any antibody or functional fragment thereof described or otherwise contemplated herein is cultured to produce any of the antibodies or functional fragments thereof described above. In at least certain non-limiting embodiments, the antibody or functional fragment thereof is recovered.

[0127] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any antibody or functional fragment thereof disclosed herein or otherwise contemplated attached to a detectable marker. Non-limiting examples of detectable markers that can be utilized according to the present disclosure include enzyme markers, radioactive markers, fluorescent markers, chemiluminescent markers, bioluminescent markers, and particulate markers, and any combination thereof. In addition, the detectable marker can be attached to the antibody or functional fragment via direct or indirect conjugation.

[0128] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any antibody or functional fragment thereof disclosed herein or otherwise contemplated attached to a solid support. The attachment of the antibody / functional fragment to a solid support (via direct or indirect conjugation) produces an affinity purification chromatography matrix, such as, for example, a column.

[0129] Certain non-limiting embodiments of the present disclosure relate to polynucleotides encoding any antibody or functional fragment thereof disclosed or otherwise contemplated herein.

[0130] In a specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain of the antibody or a functional fragment thereof has at least about 70% identity with SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 (and / or a portion of the polynucleotide encoding the heavy chain variable region of the antibody or a functional fragment thereof has at least about 70% identity with SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259), such as (but not limited to) SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 and / or SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0131] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, a portion of the polynucleotide encoding the light chain of the antibody or its functional fragment has at least about 70% identity with SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 (and / or a portion of the polynucleotide encoding the light chain variable region of the antibody or its functional fragment has at least about 70% identity with SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260), such as (but not limited to) SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 and / or SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 has at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity.

[0132] In yet another specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain of the antibody or its functional fragment has at least about 90% identity with SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249, and / or a portion of the polynucleotide encoding the light chain of the antibody or its functional fragment has at least about 90% identity with SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253.

[0133] In yet another specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain variable region of the antibody or its functional fragment has at least about 90% identity with SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259, and / or a portion of the polynucleotide encoding the light chain variable region of the antibody or its functional fragment has at least about 90% identity with SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260.

[0134] In another specific (but non-limiting) embodiment, a portion of the sequence corresponding to the heavy chain of the antibody or its functional fragment and / or a portion of the sequence corresponding to the light chain of the antibody or its functional fragment differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 or SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253, respectively, in that (and / or a portion of the sequence corresponding to the heavy chain variable region of the antibody or its functional fragment and / or a portion of the sequence corresponding to the light chain variable region of the antibody or its functional fragment, respectively, differs from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 or SEQ ID NO: ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 differs in), less than about 100 nucleotides, less than about 90 nucleotides, less than about 80 nucleotides, less than about 75 nucleotides, less than about 70 nucleotides, less than about 60 nucleotides, less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, less than about 35 nucleotides, less than about 30 nucleotides, less than about 25 nucleotides, less than about 20 nucleotides, less than about 15 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, less than about 7 nucleotides, less than about 6 nucleotides, less than about 5 nucleotides, less than about 4 nucleotides, less than about 3 nucleotides, less than about 2 nucleotides, or less than about 1 nucleotide.

[0135] In yet another specific (but non-limiting) embodiment, a portion of the sequence corresponding to the heavy chain of the antibody or its functional fragment differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229 or 249 in less than about 100 nucleotides, and / or a portion of the sequence corresponding to the light chain of the antibody or its functional fragment differs from SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233 or 253 in less than about 70 nucleotides.

[0136] In yet another specific (but non-limiting) embodiment, a portion of the sequence corresponding to the heavy chain variable region of the antibody or its functional fragment differs from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239 or 259 by less than about 35 nucleotides, and / or a portion of the sequence corresponding to the light chain variable region of the antibody or its functional fragment differs from SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 by less than about 34 nucleotides.

[0137] Certain non-limiting embodiments of the present disclosure relate to vectors comprising any polynucleotide encoding an antibody or functional fragment thereof described or otherwise contemplated herein.

[0138] Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any polynucleotide encoding an antibody or functional fragment thereof described or otherwise contemplated herein. Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any vector described or otherwise contemplated herein.

[0139] Certain non-limiting embodiments of the present disclosure relate to methods for producing antibodies or functional fragments thereof that can specifically bind to naloxone and / or naltrexone. The methods comprise: (a) culturing any recombinant host cell described herein or otherwise contemplated in cell culture under conditions that allow expression of the antibody or functional fragment thereof encoded by the polynucleotide; and (b) isolating the antibody or functional fragment thereof from the cell culture.

[0140] Certain non-limiting embodiments of the present disclosure relate to methods for detecting naloxone and / or naltrexone present in a biological sample. The methods comprise: contacting the biological sample with any antibody or functional fragment thereof disclosed or otherwise contemplated herein under conditions whereby an antibody / naloxone and / or antibody / naltrexone complex is formed, if naloxone and / or naltrexone, respectively, is present in the sample; and detecting any antibody / naloxone and / or antibody / naltrexone complex formed, wherein the amount of antibody / naloxone and / or antibody / naltrexone complex formed is proportional to the amount of naloxone and / or naltrexone, respectively, present in the sample.

[0141] In a specific (but non-limiting) embodiment, a label as described above is attached to an antibody / functional fragment for detection of antibody / naloxone and / or antibody / naltrexone complexes.

[0142] Certain non-limiting embodiments of the present disclosure relate to methods for substantially reducing (or substantially removing) naloxone and / or naltrexone interference from an opiate assay of a biological sample. The method comprises: contacting the biological sample with one or more of any antibodies or functional fragments thereof disclosed or otherwise contemplated herein under conditions whereby antibody / naloxone and / or antibody / naltrexone complexes are formed if naloxone and / or naltrexone, respectively, is present in the sample; removing any antibody / naloxone and / or antibody / naltrexone complexes formed; and performing an opiate assay on the biological sample in which naloxone and / or naltrexone has been substantially reduced / removed. The method can be used with any opiate assay and any opiate assay format. For example (but not by way of limitation), the opiate to be assayed can be codeine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone, heroin (6-acetylmorphine), combinations thereof, and the like. Non-limiting examples of opiate assay formats include ELISA, chip assays, LC / MS / MS, immunoassays, enzyme immunoassays, enzyme multiplication immunoassays, fluorescence polarization immunoassays, combinations thereof, and the like. Various types of opiate assays are well known and well used in the art. Therefore, further description thereof is deemed unnecessary to allow one of ordinary skill in the art to perform the methods disclosed herein.

[0143] In a specific (but non-limiting) embodiment, the at least one antibody or functional fragment thereof used in the method for substantially reducing (or substantially eliminating) naloxone and / or naltrexone interference from an opiate assay comprises at least two antibodies or functional fragments thereof: (1) at least one antibody or functional fragment thereof that specifically binds to naloxone, and (2) at least one antibody or functional fragment thereof that specifically binds to naltrexone.

[0144] Certain non-limiting embodiments of the present disclosure also include kits containing any antibody / functional fragment and / or composition disclosed herein or otherwise contemplated (such as a composition comprising an antibody / functional fragment attached to a detectable marker or a solid support), and any other reagents that can be used in the assays / methods described herein. For example (but not by way of limitation), the kit may further include one or more components of the opiate assay described above. For example (but not by way of limitation), the kit may include an antibody or functional fragment thereof that binds to any opiate described herein or otherwise contemplated, which is used in an opiate immunoassay.

[0145] The assay components / reagents of the compositions / test kits / methods can be provided in any form that allows them to function according to the present disclosure. For example, but not by way of limitation, each reagent can be provided in liquid form and arranged in bulk and / or in a single aliquot form in the test kit. Alternatively, in a specific (but non-limiting) embodiment, one or more of the reagents can be arranged in the test kit in the form of a single aliquot of a lyophilized reagent. The use of dry reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0146] In addition to the assay components / reagents described in detail above, the kit may further contain other reagents for carrying out any specific assay described herein or otherwise considered. The nature of these additional reagents will depend on the specific assay format, and their identification is entirely within the skill of one of ordinary skill in the art; therefore, it is not considered necessary to carry out further description thereof. In addition, the components / reagents present in the kit may each be in separate containers / compartments, or the various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. In addition, the kit may include a microfluidic device in which components / reagents are arranged.

[0147] The relative amounts of the various components / reagents in the kit can vary greatly to provide a concentration of such components / reagents that significantly optimizes the reaction that needs to occur during the assay method and further significantly optimizes the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit can be provided as a dry powder (such as a lyophilized powder), and the kit may further include an excipient for dissolving the dry reagent; in this way, a reagent solution with an appropriate concentration for carrying out the method or assay according to the present disclosure can be obtained from these components. Positive and / or negative controls may also be included in the kit. In addition, the kit may further include a set of written instructions explaining how to use the kit. A kit with this property can be used in any method described herein or otherwise considered. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 Depicted are the chemical structures of naloxone and naltrexone and various opiates with which naloxone and naltrexone cross-react in various opiate assays.

[0149] Figure 2 Schematic depiction of the synthesis of naltrexone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates linked via the C-3 position.

[0150] Figure 3Schematic depiction of the synthesis of naloxone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates linked via the C-3 position.

[0151] Figure 4 Schematic depiction of the synthesis of naltrexone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates linked via the C-6 position.

[0152] Figure 5 Schematic depiction of the synthesis of naloxone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates linked via the C-6 position.

[0153] Figure 6 Schematic depicts ELISA inhibition assays against various opiates using various bleeds obtained from selected mice immunized with naloxone- or naltrexone-KLH conjugates.

[0154] Figure 7 Schematic depicts ELISA inhibition assays of five selected anti-naloxone monoclonal antibodies using eight key opioids (immunogens indicated in the lower right of the figure).

[0155] Figure 8 Graph depicts ELISA inhibition assays of eight selected anti-naltrexone monoclonal antibodies using eight key opioids (immunogens indicated in lower panel of figure).

[0156] Fig. 9 Schematic depicts a combined specific assay for naloxone and naltrexone using monoclonal antibody 179A 6H6 (generated using a naloxone-containing immunogen).

[0157] Fig.10 Schematic depicts a naloxone and naltrexone combination specific assay using monoclonal antibody 180C 2A12 (generated using a naltrexone-containing immunogen).

[0158] Fig.11 The evaluation of monoclonal antibody 180A 3D3 in an opiate enzyme multiplier immunoassay is illustrated.

[0159] Fig.12 The evaluation of monoclonal antibody 179A 6H6 in an opiate enzyme multiplier immunoassay is illustrated.

[0160] Fig.13 The evaluation of monoclonal antibody 180C 2A12 in an opiate enzyme multiplier immunoassay is illustrated. Specific implementation plan

[0161] Examples are provided below. However, it should be understood that the present disclosure and its application are not limited to the specific experiments, results and laboratory procedures disclosed herein. On the contrary, the examples are provided only as one of the various embodiments and are intended to be exemplary, not exhaustive.

[0162] Example 1: Generation of Immunogens Comprising Naloxone and Naltrexone Conjugates This example illustrates the generation of various conjugates containing naloxone or naltrexone via multiple C-position attachments.

[0163] Materials and Equipment: Compounds were purified on a Shimadzu HPLC system equipped with a silica-bonded-C18 reverse phase column. Chemical reactions were monitored by TLC (thin layer chromatography) using silica gel plates from Analtech Inc. (Newark, DE). Silica gel plates were visualized using UV shortwave (254 nm). All chemicals were obtained from Sigma Aldrich (St.Louis, MO), Fluka (Waltham, MA), Thermo Scientific (Waltham, MA) and VWR (Radnor, PA) and used as is. Data were recorded on a Bruker ULTRASHIELD™ 600 MHz spectrometer (Bruker, Billerica, MA). 1 HNMR.Chemical shifts are reported in parts per million (ppm, δ) and are associated with tetramethylsilane or with deuterated solvents as internal references.The NMR abbreviations used are: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), dt (double triplets), td (three doublets), ddd (double double doublet), J (coupling constant), Hz (hertz).ESI-MS spectra were recorded on an Agilent HPLC1290 single quadrupole system equipped with a 6130 mass detector at Siemens Healthineers analytical facility (Newark, DE).UV: Carry 60 was used for OD280 and BCA concentration measurements.

[0164] The following abbreviations have the meanings set out below: BCA Assay - Bicinchoninic Acid Assay eq. - molar equivalent g - grams mg - milligrams mmol, mM - millimole nm - nanometer CV - Column Volume cm 2 - Square centimeters DCM - Dichloromethane MeOH - Methanol MeOD- d 4 - Deuterated methanol with 4 deuterium atoms (for NMR spectroscopy) DMF - N,N-dimethylformamide SuOH - N-Hydroxysuccinimide NaOAc - Sodium Acetate AcOH - acetic acid Ao - - Acetate EDAC∙HCl – N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride TFA - Trifluoroacetic acid TLC - Thin Layer Chromatography Rf - Retention Factor in TLC Analysis UV - Ultraviolet v / v - volume ratio ESI-MS - Electrospray Ionization Mass Spectrometry m / z - Mass-to-charge ratio NMR - Nuclear Magnetic Resonance MHz - Megahertz nG6PDH - Natural Glucose-6-Phosphate Dehydrogenase β-NADH - Nicotinamide adenine dinucleotide G6PDNa2- Glucose-6-phosphate disodium salt OVA - Ovalbumin isolated from chicken egg white mcKLH - Mariculture Keyhole Hemocyanin (ThermoFisher Scientific) TRIS - Tris(hydroxymethyl)aminomethane Dimeric protein complex of KU-nG6PDH RPM - Revolutions per minute CFA - Complete Freund's Adjuvant IFA - Incomplete Freund's Adjuvant ELISA - Enzyme-Linked Immunosorbent Assay EIA - Enzyme Immunoassay PBS - Phosphate Buffered Saline HRP - Horseradish Peroxidase TMB - 3,3',5,5'-Tetramethylbenzidine µL - microliter µg - microgram V H - Variable heavy chain V L - Variable light chain C H – Constant heavy chain C L - Constant light chain NAD + - Oxidized Nicotinamide Adenine Dinucleotide NADH - reduced nicotinamide adenine dinucleotide Figure 2 The synthesis of the naltrexone hapten, KLH immunogen, and G6PDH conjugate linked via the C-3 position is schematically depicted, as described in further detail below.

[0165] Preparation of compound (2): In an oven-dried 50 mL round-bottom flask equipped with a magnetic stir bar, naltrexone hydrochloride (1) (330 mg, 0.87 mmol) was added to the flask, followed by DMF (5 mL), acetone (5 mL) and potassium carbonate (575 mg, 4.16 mmol, 4.78 eq.). The resulting reaction mixture was stirred at room temperature (rt.) for 5 minutes under a stream of nitrogen; then, ethyl-5-bromovaleric acid (375 µL, 495 mg, 2.36 mmol, ~2.71 eq.) was added to the mixture. The resulting reaction mixture was placed in an oil bath and a condenser was attached to the round-bottom flask. The reaction mixture was heated at 60°C with stirring and after 4 hours by TLC [(DCM / MeOH 8 / 2 v / v) Rf (1) = 0.36, Rf (2) = 0.80] to complete the reaction. The solvent was removed on a rotary evaporator to obtain a white precipitate, which was suspended in acetonitrile (2 x 10 mL) and filtered out. The organic extract was concentrated on a rotary evaporator and further dried overnight (16 h) on an oil pump to remove trace volatiles. The product was suspended in acetonitrile / water (30 / 70 v / v, containing 0.1% acetic acid) and injected into a Shimadzu HPLC system for purification. The fractions containing the desired product were combined, concentrated on a rotary evaporator, and lyophilized overnight (16-20 hours) to obtain 474.8 mg of compound (2) as a colorless powder with a yield of 93%. Compound (2): ESI-MS m / z :[C 27 H 36 NO6] + Calculated value 470.3, found value [M+H]+ = 470.3; 1 H NMR (600 MHz, MeOD- d 4 ) δ: 6.86(d, J = 8.28 Hz, 1H), 6.81 (d, J = 8.28 Hz, 1H), 4.97 (br s, 1H), 4.90 (s, 1H), 4.18-4.16 (m, 2H), 4.11 (q, J = 7.14 Hz, 3H), 3.45 (d, J = 19.74 Hz, 1H), 3.40(dd, J = 13.62; 7.20 Hz, 1H), 3.25 (dd, J = 12.69, 4.35 Hz, 1H), 3.19 (dd, J =19.77, 6.27 Hz, 1H), 3.08 – 3.01 (m, 2H), 2.84 (td, J = 13.28, 4.68 Hz, 1H),2.74 (td, J = 12.93, 3.86 Hz, 1H), 2.41 – 2.38 (m, 2H), 2.25 (dt, J = 14.64, 3.00Hz, 1H), 2.12 – 2.09 (m, 1H), 1.78 – 1.74 (m, 4H), 1.72 – 1.68 (m, 2H), 1.24(t, J = 7.14 Hz, 3H), 1.18 – 1.15 (m, 1H), 0.86 – 0.83 (m, 1H), 0.79 – 0.75 (m,1H), 0.59 – 0.50 (m, 2H).

[0166] Preparation of compound (3): Compound (2) (474.8 mg, 0.81 mmol) was dissolved in MeOH (5 mL); then, sodium hydroxide 10N solution (0.53 mL) was added and the resulting reaction mixture was stirred at room temperature for 2 hours. By TLC [(DCM / MeOH 8 / 2 v / v), R (2) = 0.80 Rf (3)= 0.20] check to confirm that the reaction is complete. The reaction mixture was acidified with HCl (12N, 0.5 mL) on an ice bath. The solvent was removed on a rotary evaporator. The resulting white filter cake was suspended in acetonitrile (2 mL) and filtered out. The filtrate was concentrated on a rotary evaporator. Then water containing 0.1% TFA was added, and the resulting sample was freeze-dried overnight (16-20 hours). After freeze-drying, 471 mg of the title compound was obtained as a colorless powder with a 100% yield. Compound (3): ESI-MS m / z :[C 25 H 32 NO6] + Calculated value 442.2, found value [M+H] + = 442.2; 1 H NMR (600 MHz, MeOD- d 4 ) δ: 6.85 (d, J = 8.28 Hz, 1H), 6.78 (d, J = 8.28 Hz, 1H), 4.86 (s, 1H), 4.20 – 4.18 (m, 2H), 3.91 (d, J = 6.06 Hz, 1H), 3.37 (d, J = 19.54 Hz, 1H), 3.19(dd, J = 13.32, 7.08 Hz, 1H), 3.11 (dd, J = 12.66, 4.74 Hz, 1H), 3.07 – 3.06 (m,1H), 3.02 (dd, J = 14.55, 5.13 Hz, 1H), 2.89 (dd, J = 13.38, 7.32 Hz, 1H), 2.76(td, J = 13.16, 4.90 Hz, 1H), 2.61 (td, J = 12.82, 4.00 Hz, 1H), 2.34 – 2.32 (m,2H), 2.26 (dt, J = 14.61, 3.14 Hz, 1H), 2.04 – 2.02 (m, 1H), 1.81 – 1.76 (m,4H), 1.70 (td, J = 14.31, 3.52 Hz, 1H), 1.65 (dd, J= 13.44, 3.00 Hz, 1H), 1.28 –1.10 (m, 1H), 0.81 – 0.78 (m, 1H), 0.75 – 0.71 (m, 1H), 0.51 – 0.44(m, 2H).

[0167] Preparation of compound (4): Compound (3) (10.6 mg, 0.019 mmol) was dissolved in degassed DMF (1.06 mL) to prepare a 10 mg / mL hapten-DMF solution; then, H-hydroxysuccinimide (6.58 mg, 0.057 mmol, 3 eq.) was added. The resulting reaction mixture was stirred until homogeneous, and then EDAC∙HCl (7.30 mg, 0.038 mmol, 2 eq.) was added. The resulting reaction mixture was stirred at room temperature for 20 hours and analyzed by TLC [(DCM / MeOH 8 / 2 v / v), Rf (3) = 0.80, Rf (4) = 0.20)] monitoring. The formation of compound (4) was confirmed by TLC and MS direct loop: ESI m / z :[C 29 H 35 N2O8] + Calculated = 539.2, found [M+H] + = 539.3 and [M+Na] + = 561.3. This compound (4) was used in the next reaction without any further purification.

[0168] Preparation of naltrexone OVA (5): OVA, (12 mg, 2.81x10 -4 mmol) was dissolved in 50 mM phosphate buffer, pH 7.41 (2 mL). The resulting protein solution was gently vortexed until homogeneous; then, it was cooled to 0-4°C using an ice bath. The previously prepared compound (4)-DMF solution (0.501 mL, 40 eq.) was added dropwise to the protein solution under gentle vortexing. The resulting reaction mixture was shaken at 4°C for 20 hours (overnight) and then filtered on a G-25M SEPHADEX® column (GE Healthcare, Chicago, IL) (CV = 70 cm 3 , pre-equilibrated with 2 CV of 50 mM phosphate buffer, pH 7.00. 280 The elution of the conjugate was monitored at OD 100. A peak containing the desired naltrexone-OVA conjugate was eluted in the interval 67-90 mL. The naltrexone-OVA (5) was collected and concentrated. The concentration of the conjugate was measured by OD280 Determined to be 0.89 mg / ml. A total of 9 mg of OVA conjugate (5) was obtained with a yield of 75%.

[0169] Preparation of Naltrexone KLH Immunogen (6): Naltrexone KLH immunogen (6) was prepared using the same protocol as described for the preparation of Naltrexone OVA (5). In this case, 20 mg of mcKLH was used, which was diluted with Compound (4)-DMF solution (0.488 mL, 7 x 10 -3 mmol). After purification, the concentration of the conjugate was determined by BCA assay to be 0.96 mg / ml. 15.35 mg of KLH immunogen (6) was obtained with a yield of 76.8%.

[0170] Preparation of native G6PDH for bioconjugation: The native G6PDH enzyme emulsion (5.5 mL, 5.5 KU, 64.35 mg) was centrifuged at 18,000 RPM for 30 minutes at 4°C. The resulting clear aqueous layer was discarded, and the remaining white cake was suspended in 55mM TRIS buffer pH 8.00 (2.00 mL). The suspension was gently vortexed until dissolved; then, it was loaded onto a pre-equilibrated G-25M Sephadex column (CV = 196 mL) with 55mM TRIS buffer pH 8.00. The enzyme was eluted in intervals of 75–116 mL. The concentration of the enzyme was adjusted to 5 mg / mL using an Amicon stirred cell concentrator (30,000 MW cutoff); then, it was cooled to 0-4°C using an ice bath. Glucose-6-phosphate disodium salt [G6PDNa2] 240 mg was then added. The resulting reaction mixture was gently vortexed for 1 minute; then, β-NADH (120 mg) was added and the resulting reaction mixture was stirred again for 1 minute. The enzyme solution was aliquoted into three plastic tubes (1.90 mL corresponding to 9.50 mg of enzyme per tube). Tubes 1-3 containing native G6PDH were stored on an ice bath and prepared for compound (4) addition.

[0171] Preparation of compound (4) for bioconjugation: Compound (3) (9.71 mg, 0.017 mmol) was dissolved in degassed DMF (0.242 mL) to prepare a 40 mg / mL hapten-DMF solution; then, N-hydroxysuccinimide (6.20 mg, 0.054 mmol, ~3 eq.). The resulting reaction mixture was stirred until homogeneous, and then EDAC∙HCl (7.30 mg, 0.038 mmol, 2 eq.) was added. The resulting reaction mixture was stirred at room temperature for 22 hours and analyzed by TLC [(DCM / MeOH 8 / 2 v / v), Rf (4) = 0.80, Rf (3) = 0.20] monitoring. After the formation of compound (4), degassed DMF (0.242 μL) was added to prepare a 20 mg / mL compound (4)-DMF solution. ESI m / z :[C 29 H 35 N2O8] + Calculated value = 539.3, found value [M+H] + = 539.3, [M+Na] + = 561.3; this compound (4) was used for bioconjugation without any further purification.

[0172] Preparation of naltrexone nG6PDH enzyme (7a-c): In tube 1, compound (4) (243 μL - DMF solution) [10-fold molar excess of compound (4) to nG6PDH]. The reaction mixture was shaken at 2-8°C for 1 hour; then, it was quenched with L-lysine [1M solution, 9 μL, 10-fold molar excess of L-lysine to compound (4)]. The reaction mixture was then shaken at room temperature for 20 minutes. The conjugate was passed through a G-50M SEPHADEX pre-equilibrated with 55 mM TRIS buffer pH 7.00 (2 CV). ® Column (CV = 67 cm 2 ) was purified. The conjugate eluted as a sharp peak in the interval of 45-60 mL. All fractions containing the desired conjugate were collected and concentrated. The concentration of the conjugate was determined by OD 280 The concentration was determined to be 1.16 mg / mL. G6PDH conjugate (7a) was obtained at 7.93 mg with a yield of 83%.

[0173] In tube 2, compound (4) (485 μL - DMF solution) (10-fold molar excess of compound (4) to nG6PDH) was added; the reaction mixture was shaken at 2-8°C for 1 hour; then, it was quenched with L-lysine [1M solution, 18 μL, 10-fold molar excess of L-lysine to compound (4)]. The reaction mixture was then shaken at room temperature for 20 minutes. The conjugate was passed through G-50M SEPHADEX pre-equilibrated with 55mM TRIS buffer pH7.00 (2 CV) ® Column (CV = 67 cm 2 ) was purified. The conjugate eluted as a sharp peak in the interval of 45-60 mL. All fractions containing the desired conjugate were collected and concentrated. The concentration of the conjugate was determined by OD 280 The concentration was determined to be 1.25 mg / mL. G6PDH conjugate (7b) was obtained at 8.09 mg with a yield of 85%.

[0174] In tube 3, compound (4) (729 μL - DMF solution) [compound (4) 30-fold molar excess to nG6PDH] was added. The reaction mixture was shaken at 2-8°C for 1 hour; then, it was quenched with L-lysine [1M solution, 27 μL, L-lysine 10-fold molar excess to compound (4)]. The reaction mixture was then shaken at room temperature for 20 minutes. The conjugate was passed through G-50M SEPHADEX pre-equilibrated with 55 mM TRIS buffer pH 7.00 (2 CV). ® Column (CV = 67 cm 2 ) was purified. The conjugate eluted as a sharp peak in the interval of 45-60 mL. All fractions containing the desired conjugate were collected and concentrated. The concentration of the conjugate was determined by OD 280 The concentration was determined to be 1.28 mg / mL. G6PDH conjugate (7c) was obtained at 8.27 mg with a yield of 87%.

[0175] Figure 3 The synthesis of the naloxone hapten, KLH immunogen, and G6PDH conjugate linked via the C-3 position is schematically depicted, as described in further detail below.

[0176] Preparation of Compound (9): This compound was prepared using the same synthetic method as described for the preparation of Compound (2). After freeze drying, Compound (9) (465.5 mg) was obtained as a colorless powder in 99% yield. Compound (9): ESI-MS m / z :[C 26 H 34 NO6] + Calculated value 456.2, found value [M+H]+ = 456.2; 1 H NMR (600 MHz, MeOD- d 4 ) δ: 6.87(d, J = 8.28 Hz, 1H), 6.82 (d, J = 8.28 Hz, 1H), 6.03 – 5.96 (m, 1H), 5.71 (d, J =16.98 Hz, 1H), 5.64 (d, J = 10.26 Hz, 1H), 4.90 (s, 1H), 4.20 – 4.16 (m, 2H), 4.11 (q, J = 7.14 Hz, 2H), 3.98 (dd, J = 13.59, 8.62 Hz, 1H), 3.89 (dd, J = 13.62,5.64 Hz, 1H), 3.78 (d, J = 6.00 Hz, 1H), 3.48 (d, J = 19.80 Hz, 1H), 3.31 – 3.28(m, 1H), 3.10 (dd, J = 19.80, 6.18 Hz, 1H), 3.03 (td, J = 14.65, 4.97 Hz, 1H), 2.87 – 2.75 (m, 2H), 2.40 (m, 2H), 2.24 (dt, J = 14.70, 3.00 Hz, 1H), 2.06 –2.02 (m, 1H), 1.78 – 1.76 (m, 4H), 1.73 – 1.66 (m, 2H), 1.25 (t, J = 7.14 Hz, 3H).

[0177] Preparation of Compound (10): This compound was prepared using the same synthetic method as described for the preparation of Compound (3). After freeze drying, Compound (10) (447 mg) was obtained as a colorless powder in an almost 100% yield. Compound (10): ESI-MS m / z :[C 24 H 30 NO6] + Calculated = 428.2, found [M+H] + = 428.2;1 H NMR (600 MHz, MeOD- d 4 )δ: 6.82 (d, J = 8.22 Hz, 1H), 6.75 (d, J = 8.22 Hz, 1H), 5.99 – 5.91 (m, 1H), 5.48(dd, J = 17.13, 1.17 Hz, 1H), 5.43 – 5.41 (m, 2H), 4.82 (s 1H), 4.18 – 4.16 (m, 2H), 3.60 (dd, J = 13.60, 7.53 Hz, 1H), 3.55 (dd, J = 13.59, 6.03 Hz, 1H), 3.42(d, J = 5.88 Hz, 1H), 3.30 (s, 1H), 3.03 (td, J = 14.55, 5.10 Hz, 1H), 2.96 (dd, J = 12.48, 4.80 Hz, 1H), 2.85 (dd, J = 19.32, 6.06 Hz, 1H), 2.65 (td, J = 12.99,5.04 Hz, 1H), 2.46 (td, J = 12.63, 3.92 Hz, 1H), 2.36 – 2.33 (m, 2H), 2.22 (dt, J = 14.52, 3.12 Hz, 1H), 1.97-1.94 (m, 1H), 1.80-1.76 (m, 4H), 1.65 (dd, J =14.25, 3.45 Hz, 1H), 1.61-1.58 (m, 1H).

[0178] Preparation of compound (11): This compound was prepared using the same synthetic method as described for compound (4).

[0179] Preparation of naloxone OVA (12): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0180] Preparation of Naloxone KLH Immunogen (13): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0181] Preparation of naloxone nG6PDH [14(ac)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDHs [7(ac)].

[0182] Figure 4 The synthesis of the naltrexone hapten, KLH immunogen, and G6PDH conjugate linked via the C-6 position is schematically depicted, as described in detail below.

[0183] Preparation of compound (15): Naltrexone hydrochloride (1) (118 mg, 0.31 mmol) was dissolved in MeOH (7 mL); then, NaOAc (292 mg, 2.24 mmol) was added. The resulting reaction mixture was stirred at room temperature for 5 minutes; then, O-(carboxymethyl)hydroxylamine hemihydrochloride (210.7 mg, 1.92 mmol) was added to the mixture. The resulting reaction mixture was stirred at room temperature overnight (20 hours). TLC analysis of the reaction mixture showed that the reaction was complete [(DCM / MeOH 8 / 2 v / v)R f(1) = 0.4, R f(15) = 0.02]. The solvent was removed on a rotary evaporator to give a white suspension. Then, water / acetonitrile (1.4 mL / 0.6 mL v / v) containing 0.1% AcOH was added. The crude reaction mixture was then injected into a preparative Shimadzu HPLC system for purification. The fractions containing the desired product were combined, concentrated on a rotary evaporator, and lyophilized overnight (16-20 hours). This gave compound (15) (124 mg) as a colorless powder in 75% yield. Compound (15): ESI-MS m / z: [C 22 H 27 NO6] + Calculated value 415.2, found value [M+H] + = 415.2, and [M+Na] + = 429.2; 1 H NMR (600 MHz, MeOD- d 4 ) δ: 6.76 (d, J = 8.10 Hz, 1H), 6.72 (d, J = 8.22 Hz, 1H), 5.06(s, 1H), 4.66 (s, 2H), 3.99 (d, J = 6.60 Hz, 1H), 3.42 (d, J = 19.74 Hz, 1H),3.38 (dd,J = 13.32, 7.03 Hz, 1H), 3.17 (dd, J =12.99, 5.01 Hz, 1H), 3.12 (dd, J =19.86, 6.84 Hz, 1H), 2.97 (dd, J = 13.62, 7.50 Hz, 1H), 2.91 (td, J = 13.05, 4.04Hz, 1H), 2.82 (ddd, J = 17.76, 6.54, 1.86 Hz, 1H), 2.68 (td, J = 13.43, 5.00 Hz,1H), 2.65 – 2.59 (m, 1H), 1.80 – 1.76 (m, 2H), 1.51 (ddd, J = 14.25, 12.09,6.54 Hz, 1H), 1.17 – 1.10 (m, 1H), 0.88 – 0.84 (m, 1H), 0.80 – 0.75 (m, 1H), 0.57 – 0.50 (m, 2H).

[0184] Preparation of compound (16): This compound was prepared using the same synthetic method as described for compound (4).

[0185] Preparation of Naltrexone OVA (17): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0186] Preparation of Naltrexone KLH Immunogen (18): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0187] Preparation of naltrexone nG6PDH [19(ac)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDHs [7(ac)].

[0188] Figure 5 The synthesis of the naloxone hapten, KLH immunogen, and G6PDH conjugate linked via the C-6 position is schematically depicted, as described in detail below.

[0189] Preparation of compound (20): Naloxone hydrochloride (8) (110 mg, 0.27 mmol) was dissolved in MeOH (7 mL); then, NaOAc (260 mg, 2.00 mmol) was added. The resulting reaction mixture was stirred at room temperature for 5 minutes; then, O -(Carboxymethyl)hydroxylamine hemihydrochloride (186 mg, 1.69 mmol). The resulting reaction mixture was stirred at room temperature overnight. TLC analysis showed that the reaction was complete (DCM / MeOH 8 / 2 v / v Rf (1) = 0.40, Rf (20) = 0.02). The reaction mixture formed a white precipitate. The reaction mixture was concentrated on a rotary evaporator to give a white suspension, which was dissolved in water containing 0.1% AcOH and injected into a Shimadzu HPLC for purification. The fractions containing the desired product were combined, concentrated on a rotary evaporator, and lyophilized overnight (16-20 hours). This gave compound (20) (125 mg) in 87% yield. Compound (20): ESI-MS m / z: [C 21 H 25 NO6] + Calculated value 401.2, found value [M+H] + = 401.2, [M+Na] + = 423.2; 1 H NMR (600 MHz, MeOD- d 4 ) δ: 6.74 (d, J = 8.17 Hz, 1H), 6.70 (d, J = 8.22 Hz,1H), 5.98 – 5.90 (m, 1H), 5.68 (d, J = 17.16 Hz, 1H), 5.64 (d, J = 10.38 Hz, 1H), 5.04 (s, 1H), 4.64 (s, 2H), 3.95 (dd, J = 13.65, 8.67 Hz, 1H), 3.81 (dd, J =13.68, 5.58 Hz, 1H), 3.63 (d, J = 6.54 Hz, 1H), 3.43 (d, J = 19.74 Hz, 1H), 3.22(dd, J= 12.93, 4.95 Hz, 1H), 3.02 (dd, J = 19.80, 6.60 Hz, 1H), 2.92 (td, J =13.13, 4.10 Hz, 1H), 2.80 (ddd, J = 17.73, 6.39, 1.89 Hz, 1H), 2.65 (td, J =13.47, 5.04 Hz, 1H), 2.61 – 2.54 (m, 1H), 1.77 (dd, J = 13.68, 3.30 Hz, 1H),1.71 (ddd, J = 14.26, 6.67, 2.38 Hz, 1H), 1.45 (ddd, J = 14.26, 12.20, 6.43 Hz, 1H).

[0190] Preparation of compound (21): This compound was prepared using the same synthetic method as described for compound (4).

[0191] Preparation of naloxone OVA (22): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0192] Preparation of Naloxone KLH Immunogen (23): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0193] Preparation of naloxone nG6PDH [24(ac)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDHs [7(ac)].

[0194] Example 2: Generation of anti-naloxone and anti-naltrexone monoclonal antibodies This example illustrates the generation of specific monoclonal antibodies against naloxone and naltrexone. These monoclonal antibodies can be used in naloxone and naltrexone specific assays, respectively.

[0195] Furthermore, these monoclonal antibodies can be used as blocking antibodies in opiate assays, such as, but not limited to, hydrocodone assays. It is often observed that various opiate assays provide false positive results for patients undergoing naloxone / naltrexone treatment. In such cases, the naloxone / naltrexone specific assays disclosed herein can be used to screen patients to exclude any false positives. Additionally (and / or alternatively), biological samples utilized in various opiate assays can be subjected to a pretreatment step with these naloxone / naltrexone specific monoclonal antibodies to remove any naloxone / naltrexone present in the sample and thereby prevent or substantially reduce any naloxone / naltrexone interference observed in quantitative clinical opiate assays.

[0196] In this example, various immunogens containing naloxone or naltrexone attached to KLH at various locations were prepared and used as immunogens to immunize mice (e.g., BALB / c mice, Swiss Webster mice, or AJ strains of mice) intraperitoneally. Mice were immunized three or more times to generate a high titer immune response. An adjuvant such as CFA (using immunogen emulsified with CFA) was used for the initial immunization, followed by an adjuvant such as IFA for subsequent booster immunizations. After immunization, mice were bled and serum samples were separated using centrifugation techniques. Serum samples from these mice were tested for anti-naloxone and anti-naltrexone antibodies using a conjugate of naloxone and / or naltrexone with ovalbumin (ovalbumin conjugate). Microtiter plate ELISA was used, and inhibition ELISA was used to examine the binding of antibodies to the ovalbumin conjugate and subsequent binding to free drug molecules. Figure 6 Inhibition ELISA results using eight important opioid drugs are shown. Figure 6 The immunogens for mouse bleeding shown in the figure are as follows. Top row, from left to right: naltrexone 3-KLH (Formula III, wherein R2 is cyclopropyl); naltrexone 6-KLH (Formula VI, wherein R2 is cyclopropyl); naloxone 3-KLH (Formula III, wherein R2 is allyl); and naloxone 3-KLH. Bottom row, from left to right: naltrexone 3-KLH; naltrexone 6-KLH; naloxone 6-KLH (Formula VI, wherein R2 is allyl); and naloxone 6-KLH.

[0197] Mice with good anti-drug antibody titers and binding to free drug in ELISA inhibition assays were selected for the generation of monoclonal antibodies in a manner similar to that described in U.S. Pat. No. 9,815,907 (granted to Sharma et al. on November 14, 2017). Specifically, mice with the highest titers and best specificity were boosted three days before fusion. On the day of fusion, splenocytes were harvested from these mice and fused with the myeloma cell line P3X63Ag8.653 using a PEG-assisted fusion protocol. After about ten days, hybridoma supernatants were screened for anti-naloxone and anti-naltrexone antibodies using plate ELISA (direct binding and inhibition ELISA for specificity). Positive clones were further amplified and subcloned, and protein A SEPHAROSE was used to detect the presence of naloxone and naltrexone antibodies. ® The supernatant was purified on a ELISA column (GE Healthcare, Chicago, IL). Purified antibody samples were tested for binding to the immunogen (ie, drug-carrier protein conjugate) and free opioid using ELISA.

[0198] Table 1 lists the names of some of the selected hybridoma clones generated against naloxone and naltrexone drug molecules, as described herein.

[0199] Table 1: List of monoclonal antibodies generated against naloxone- and naltrexone-KLH conjugates mAb name drug 179B 9G1.1 Naloxone 179B 9E12 Naloxone 179B 10A7 Naloxone 179A 6H6 Naloxone 179B 9E7 Naloxone 180A 3D3 Naltrexone 180B 13F7 Naltrexone 180C 1F8 Naltrexone 180C 7G8 Naltrexone 180C 8A10 Naltrexone 180C 9D5 Naltrexone 180C 9G8 Naltrexone 180C 2A12 Naltrexone

[0200] Antibody solutions (bleeds from immune mice or supernatants from hybridoma clones or purified antibodies) were screened using the inhibition ELISA procedure according to the following protocol. Plates were coated with 1 µg / mL of naloxone 3-ovalbumin, naloxone 6-ovalbumin, naltrexone 3-ovalbumin, or naltrexone 6-ovalbumin in phosphate-buffered saline, 50 µL per well. Plates were coated for 1 hour or more at room temperature or overnight at approximately 2°C to 8°C. Plates were then patted dry and coated with 200 µL per well of blocking buffer (containing 0.05% TWEEN ® The plates were blocked with 0.5% casein solution (Croda International PLC, Snaith, United Kingdom) in PBS containing 20 surfactant. Plate blocking was performed by incubation at room temperature with plate shaking for 30 minutes or more. The plates were washed using a plate washer (BioTek®, Winooski, VT) with a plate stacker, wherein the wash buffer was 0.05% TWEEN ® 20 of MILLIQ ®Water (Millipore Corporation, Billerica, MA). The monoclonal antibody to be screened is then added to each well along with free drug as follows: 25 µL of appropriately diluted culture supernatant (or antibody solution) per well, and 25 µL of 20 µg / mL free drug is added. Incubation is performed at room temperature with plate shaking for approximately 1 hour. The plates are washed. Enzyme conjugate (goat anti-mouse IgG conjugated to HRP diluted to 1:3000 in blocking buffer) is added, 50 µL per well. Incubation is performed at room temperature with shaking for approximately 1 hour. The plates are then washed, and the color development solution (TMB from Moss Substrates, Pasadena MD) is added in a volume of 100 µL per well. If the desired antibody is present in the hybridoma supernatant, a decrease in optical density is observed compared to wells without free drug. The plates are read at a wavelength of 650 nm using an ELISA plate reader (Molecular Devices, San Jose, CA). NOTE: The appropriate dilution of the antibody solution is determined by performing a titration ELISA. Select the antibody dilution where an optical density of approximately 1.0 is observed.

[0201] like Figure 7 As shown in , monoclonal antibodies designated 179B 9G1.1, 179B 9E12, 179B 10A7, 179A 6H6, and 179B 9E7 were generated using naloxone-3-Val-mcKLH (Formula III, wherein R2 is allyl) and had better specificity for naloxone than for eight other key opioids tested in an inhibition ELISA assay. Figure 8 As shown in , monoclonal antibodies designated 180B 13F7, 180C 1F8, 180C7G8, 180C 8A10, 180C 9D5, 180C 9G8, 180C 2A12, and 180A 3D3 were generated using naltrexone-3-Val-mcKLH (Formula III, wherein R2 is cyclopropyl) and had specificity for naltrexone better than inhibition of eight other key opioids tested in ELISA assays. Based on these results, the monoclonal antibodies were then used in naloxone / naltrexone specific immunoassays and methods for preventing or substantially reducing naloxone / naltrexone interference in other opiate assays, as described in further detail below.

[0202] Example 3: Monoclonal Antibody Sequencing Hybridoma cells generated as in Example 2 were used to sequence the heavy and light chains of various monoclonal antibodies. Sequences of different clones were aligned and consensus sequences were provided. The various sequences obtained for each monoclonal antibody are shown in Tables 2-14 below.

[0203] Table 2: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 2A12 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 1 9 Heavy chain CDR1 2 10 Heavy chain CDR2 3 11 Heavy chain CDR3 4 12 Light chain 5 13 Light chain CDR1 6 14 Light chain CDR2 7 15 Light chain CDR3 8 16 Heavy chain variable region 17 19 Light chain variable region 18 20

[0204] Table 3: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180A 3D3 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 21 29 Heavy chain CDR1 22 30 Heavy chain CDR2 23 31 Heavy chain CDR3 24 32 Light chain 25 33 Light chain CDR1 26 34 Light chain CDR2 27 35 Light chain CDR3 28 36 Heavy chain variable region 37 39 Light chain variable region 38 40

[0205] Table 4: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180B13F7 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 41 49 Heavy chain CDR1 42 50 Heavy chain CDR2 43 51 Heavy chain CDR3 44 52 Light chain 45 53 Light chain CDR1 46 54 Light chain CDR2 47 55 Light chain CDR3 48 56 Heavy chain variable region 57 59 Light chain variable region 58 60

[0206] Table 5: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 1F8 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 61 69 Heavy chain CDR1 62 70 Heavy chain CDR2 63 71 Heavy chain CDR3 64 72 Light chain 65 73 Light chain CDR1 66 74 Light chain CDR2 67 75 Light chain CDR3 68 76 Heavy chain variable region 77 79 Light chain variable region 78 80

[0207] Table 6: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 7G8 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 81 89 Heavy chain CDR1 82 90 Heavy chain CDR2 83 91 Heavy chain CDR3 84 92 Light chain 85 93 Light chain CDR1 86 94 Light chain CDR2 87 95 Light chain CDR3 88 96 Heavy chain variable region 97 99 Light chain variable region 98 100

[0208] Table 7: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 8A10 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 101 109 Heavy chain CDR1 102 110 Heavy chain CDR2 103 111 Heavy chain CDR3 104 112 Light chain 105 113 Light chain CDR1 106 114 Light chain CDR2 107 115 Light chain CDR3 108 116 Heavy chain variable region 117 119 Light chain variable region 118 120

[0209] Table 8: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 9D5 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 121 129 Heavy chain CDR1 122 130 Heavy chain CDR2 123 131 Heavy chain CDR3 124 132 Light chain 125 133 Light chain CDR1 126 134 Light chain CDR2 127 135 Light chain CDR3 128 136 Heavy chain variable region 137 139 Light chain variable region 138 140

[0210] Table 9: Sequence identifiers of various sequences of anti-naltrexone monoclonal antibody 180C 9G8 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 141 149 Heavy chain CDR1 142 150 Heavy chain CDR2 143 151 Heavy chain CDR3 144 152 Light chain 145 153 Light chain CDR1 146 154 Light chain CDR2 147 155 Light chain CDR3 148 156 Heavy chain variable region 157 159 Light chain variable region 158 160

[0211] Table 10: Sequence identifiers of various sequences of anti-naloxone monoclonal antibody 179B 9G1.1 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 161 169 Heavy chain CDR1 162 170 Heavy chain CDR2 163 171 Heavy chain CDR3 164 172 Light chain 165 173 Light chain CDR1 166 174 Light chain CDR2 167 175 Light chain CDR3 168 176 Heavy chain variable region 177 179 Light chain variable region 178 180

[0212] Table 11: Sequence identifiers of various sequences of anti-naloxone monoclonal antibody 179B 9E12 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 181 189 Heavy chain CDR1 182 190 Heavy chain CDR2 183 191 Heavy chain CDR3 184 192 Light chain 185 193 Light chain CDR1 186 194 Light chain CDR2 187 195 Light chain CDR3 188 196 Heavy chain variable region 197 199 Light chain variable region 198 200

[0213] Table 12: Sequence identifiers of various sequences of anti-naloxone monoclonal antibody 179B 10A7 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 201 209 Heavy chain CDR1 202 210 Heavy chain CDR2 203 211 Heavy chain CDR3 204 212 Light chain 205 213 Light chain CDR1 206 214 Light chain CDR2 207 215 Light chain CDR3 208 216 Heavy chain variable region 217 219 Light chain variable region 218 220

[0214] Table 13: Sequence identifiers of various sequences of anti-naloxone monoclonal antibody 179A 6H6 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 221 229 Heavy chain CDR1 222 230 Heavy chain CDR2 223 231 Heavy chain CDR3 224 232 Light chain 225 233 Light chain CDR1 226 234 Light chain CDR2 227 235 Light chain CDR3 228 236 Heavy chain variable region 237 239 Light chain variable region 238 240

[0215] Table 14: Sequence identifiers of various sequences of anti-naloxone monoclonal antibody 179B 9E7 sequence SEQ ID NO of amino acid sequence: DNA sequence SEQ ID NO: Heavy chain 241 249 Heavy chain CDR1 242 250 Heavy chain CDR2 243 251 Heavy chain CDR3 244 252 Light chain 245 253 Light chain CDR1 246 254 Light chain CDR2 247 255 Light chain CDR3 248 256 Heavy chain variable region 257 259 Light chain variable region 258 260

[0216] Example 4: Use of Monoclonal Antibodies in Naloxone and Naltrexone-Specific Assays In this example, the monoclonal antibodies generated in Example 2 were used in naloxone- and naltrexone-specific immunoassays to demonstrate the ability of these monoclonal antibodies to detect one or both drugs in a clinical immunoassay.

[0217] Fig. 9 The diagram depicts a naloxone and naltrexone specific assay using the anti-naloxone monoclonal antibody 179A 6H6. As can be seen, the anti-naloxone monoclonal antibody detects both naloxone and naltrexone with substantially the same specificity. Similar results were obtained with the anti-naloxone monoclonal antibodies 179B10A7, 179B 9G1, 179B 9E12, and 179B 9E7. Therefore, these antibodies can be used in clinical testing immunoassays for one or both drugs.

[0218] Fig.10 The diagram depicts a naloxone and naltrexone specific assay using the anti-naltrexone monoclonal antibody 180C 2A12. This anti-naltrexone monoclonal antibody is able to detect naloxone in addition to naltrexone, albeit with lower specificity compared to naltrexone. However, this antibody can still be used in clinical testing immunoassays for either or both drugs.

[0219] Based on these results, these antibodies can be used in the following assay structure. ® II (enzyme multiplication immunoassay, Siemens Healthineers, Newark, DE) The naloxone and / or naltrexone assay is a homogeneous enzyme immunoassay technique used to analyze specific compounds in human urine. The assay is based on competition for antibody binding sites between the drug in the sample and the drug labeled with glucose-6-phosphate dehydrogenase (rG6PDH). After binding to the antibody, the enzyme activity decreases, so the drug concentration in the sample can be measured in terms of enzyme activity. In the presence of glucose-6-phosphate (G6P), the active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH, resulting in a change in absorbance that is measured spectrophotometrically. Endogenous serum G6PDH does not interfere because the coenzyme NAD only functions with the bacterial (Leuconostoc mesenteroides) enzyme used in the assay.

[0220] EMIT ® The assay format includes an antibody / substrate reagent, referred to as Reagent 1. This reagent includes the antibody as well as buffers, preservatives, and stabilizers for the assay.® The assay format also includes an enzyme reagent, referred to as Reagent 2. This reagent includes the conjugate as well as buffers, preservatives, and stabilizers for the assay.

[0221] Example 5: Reducing Naloxone / Naltrexone Interference in Opioid Assays In this example, the monoclonal antibodies generated in Example 2 were used in opiate enzyme multiplex immunoassays to demonstrate the ability to reduce naloxone / naltrexone interference in these assays.

[0222] Fig.11 showed that in the presence of the naltrexone antibody blocker 3D3, the cross-reactivity of naloxone and naltrexone changed from a positive response to the cutoff value (300 ng / mL) to a negative response. Fig.12 showed that in the presence of the naloxone antibody blocker 6H6, the cross-reactivity of naloxone changed from a positive response to the cutoff value (300 ng / mL) to a negative response only at much lower concentrations.

[0223] exist Fig.13 In the present study, the anti-naltrexone monoclonal antibody 2A12 was used as a blocking agent in the hydrocodone assay to increase the amount of naloxone and naltrexone that produced a negative response to the cutoff value (300 ng / ml).

[0224] Therefore, according to the present disclosure, compositions and methods of producing and using the same have been provided that fully satisfy the objects and advantages described above. Although the present disclosure has been described in conjunction with the specific drawings, experiments, results and language set forth above, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such substitutions, modifications and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. An antibody or a functional fragment thereof that can specifically bind to naloxone and / or naltrexone, wherein the antibody or functional fragment is produced by immunizing a non-human animal with a structure of any one of Formulae III-VIII: in, In Formula III: A is a heteroatom selected from -O-, -N- or -S-; B is selected from -CO-, a lower alkyl group (C0 to C 10 ), -CONH-, -SO2-, -PO4-; C is a carrier protein; and R2 is H, alkyl, allyl or methylenecyclopropyl; Wherein, in Formula IV: D is a heteroatom selected from O-, S-, CO- or CH2-; E is a lower alkyl, CO2-, CONH-, SO2- or PO4-; F is a carrier protein; R2 is H, alkyl, allyl or methylenecyclopropyl; and R1 is H or CH3; Wherein, in Formula V: D is a heteroatom selected from O-, S-, CO- or CH2-; E is a lower alkyl, CO2-, CONH-, SO2- or PO4-; F is a carrier protein; R2 is H, alkyl, allyl or methylenecyclopropyl; and R1 is H or CH3; Wherein, in Formula VI: G is a linking group, which includes at least two of the following: a lower alkyl chain, CO2-, CONH-, SO2- and PO2-; H is a carrier protein; R1 is H or CH3; and R2 is H, an alkyl group, an allyl group or a methylene cyclopropyl group; Wherein, in Formula VII: I is a heteroatom selected from O-, S-, CO- or CH2-; J is a lower alkyl chain, CO2-, CONH-, SO2- or PO4-; K is a carrier protein; R1 is H or CH3; and R2 is H, alkyl, allyl or methylenecyclopropyl; and Wherein, in Formula VII: L is a heteroatom selected from O, S, CO or CH2; M is a lower alkyl chain, CO2-, CONH-, SO2- or PO4-; N is a carrier protein; R1 is H or CH3; and R2 is H, alkyl, allyl or methylenecyclopropyl.

2. An antibody or a functional fragment thereof that can specifically bind to naloxone and / or naltrexone, wherein (1) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 9, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 13; (2) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:29, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:33; (3) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:49, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:53; (4) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:69, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:73; (5) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 89, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 93; (6) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 109, and / or the light chain is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 113; (7) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 129, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 133; (8) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 149, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 153; (9) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 169, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 173; (10) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 189, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 193; (11) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 209, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 213; (12) the heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 229, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 233; or (13) The heavy chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO: 249, and / or the light chain of the antibody is encoded by a polynucleotide sequence that is at least about 90% identical to SEQ ID NO:

253.

3. A method for producing an antibody or a functional fragment thereof that can specifically bind to naloxone, the method comprising the following steps: immunizing a non-human animal with a conjugate comprising naloxone attached to a carrier protein, thereby inducing B-cells to produce antibodies that bind to the conjugate; and The antibody or functional fragment thereof produced by the B-cell is obtained.

4. The method of claim 3, wherein the carrier protein is selected from the group consisting of KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), ovalbumin, BTG (bovine thyroglobulin) and BGG (bovine gamma globulin), and combinations thereof.

5. The method of claim 3, wherein the carrier protein is attached to the 1-carbon, 2-carbon, 3-carbon, 6-carbon, 7-carbon or 8-carbon position of naloxone.

6. A method for producing an antibody or a functional fragment thereof that can specifically bind to naltrexone, the method comprising the following steps: immunizing a non-human animal with a conjugate comprising naltrexone attached to a carrier protein, thereby inducing B-cells to produce antibodies that bind to the conjugate; and The antibody or functional fragment thereof produced by the B-cell is obtained.

7. The method of claim 6, wherein the carrier protein is selected from the group consisting of KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), ovalbumin, BTG (bovine thyroglobulin) and BGG (bovine gamma globulin), and combinations thereof.

8. The method of claim 6, wherein the carrier protein is attached to the 1-carbon, 2-carbon, 3-carbon, 6-carbon, 7-carbon, or 8-carbon position of naltrexone.

9. A hybridoma producing the antibody or a functional fragment thereof according to claim 1 or 2.

10. A method for producing an antibody or a functional fragment thereof that can specifically bind to naloxone and / or naltrexone, the method comprising: Cultivating the hybridoma according to claim 9 to produce the antibody or a functional fragment thereof; and Recovering the antibody or functional fragment thereof.

11. A composition comprising: At least one antibody or a functional fragment thereof according to claim 1 or 2; and A detectable label is attached to the at least one antibody or functional fragment thereof.

12. A composition comprising: a solid support; and At least one antibody or a functional fragment thereof according to claim 1 or 2 bound to the solid support.

13. A polynucleotide encoding the antibody or a functional fragment thereof according to claim 1 or 2.

14. A vector comprising a polynucleotide encoding the antibody or a functional fragment thereof according to claim 1 or 2.

15. A recombinant host cell comprising a polynucleotide encoding the antibody or a functional fragment thereof according to claim 1 or 2.

16. A method for producing an antibody or a functional fragment thereof that can specifically bind to naloxone and / or naltrexone, the method comprising: (a) cultivating the recombinant host cell of claim 15 in cell culture under conditions that allow expression of the antibody or functional fragment thereof encoded by the polynucleotide; and (b) isolating the antibody or a functional fragment thereof from the cell culture.

17. A method for detecting the presence of naloxone and / or naltrexone in a biological sample, the method comprising the steps of: contacting the biological sample with the antibody or functional fragment thereof according to claim 1 or 2 under conditions whereby an antibody / naloxone and / or antibody / naltrexone complex is formed, if naloxone and / or naltrexone, respectively, is present in the biological sample; and Any antibody / naloxone and / or antibody / naltrexone complexes formed are detected, wherein the amount of antibody / naloxone and / or antibody / naltrexone complexes formed is proportional to the amount of naloxone and / or naltrexone present in the biological sample.

18. The method of claim 17, wherein the antibody or functional fragment thereof has a label attached thereto, said label being used to detect the antibody / naloxone and / or antibody / naltrexone complex.

19. A method for substantially reducing interference from naloxone and / or naltrexone in at least one opiate assay in a biological sample, the method comprising the steps of: contacting the biological sample with at least one antibody or a functional fragment thereof according to claim 1 or 2 under conditions whereby an antibody / naloxone and / or antibody / naltrexone complex is formed, if naloxone and / or naltrexone, respectively, is present in the sample; removing any antibody / naloxone and / or antibody / naltrexone complexes that have formed; and At least one opiate assay is performed on the biological sample in which naloxone and / or naltrexone has been substantially reduced.

20. The method of claim 19, wherein the opiate to be assayed is selected from the group consisting of codeine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone, heroin (6-acetylmorphine), and combinations thereof.

21. The method of claim 19, wherein the opiate assay is selected from the group consisting of ELISA, chip assay, LC / MS / MS, immunoassay, enzyme immunoassay, enzyme multiplication immunoassay, fluorescence polarization immunoassay, and combinations thereof.

22. The method of claim 19, wherein the at least one antibody or functional fragment thereof comprises at least one antibody or functional fragment thereof that specifically binds to naloxone and at least one antibody or functional fragment thereof that specifically binds to naltrexone.

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