Anti-naloxone and anti-naltrexone monoclonal antibodies and methods for making and using same

Abstract

To provide antibodies or functional fragments thereof which can specifically bind to naloxone and / or naltrexone, and methods of production thereof.SOLUTION: The invention provides: antibodies or functional fragments thereof which include heavy chain variable regions CDR1, CDR2, and CDR3 each having a specific amino acid sequence and light chain variable regions CDR1, CDR2, and CDR3 each having a specific amino acid sequence; methods of production of the antibodies or functional fragments thereof; and compositions comprising the antibodies or functional fragments thereof and detectable labels attached thereto.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS / INCORPORATION-BY-REFERENCE STATES Not applicable

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable [Background technology]

[0003] Naloxone is commonly used as an antagonist of abused opioids, typically in cases of drug overdose. Naloxone is a life-saving, short-acting medication that reverses an opiate overdose by removing the opiate from the opiate receptors. Patients are given repeated injections of naloxone (NARCAN® (naloxone HCl), ADAPT Pharma, Inc., Radnor, PA.) to saturate the drug receptors, thereby minimizing the pharmacological response to the abused opioid. Naloxone acts within minutes and lasts for approximately one hour.

[0004] Naltrexone is another commonly used opioid antagonist that is also used in cases of alcoholism; naltrexone is an opiate / alcohol blocker that has been used for the past 30 years. Naltrexone acts slowly and lasts longer than naloxone. VIVITROL® (Alkermes, Inc., Dublin, Ireland) is a sustained-release form of naltrexone. The sustained-release properties of VIVITROL® aid in patient compliance, as daily use is not required.

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

[0006] Naloxone has rapid systemic distribution. Its mean serum half-life has been shown to range from 30 to 81 minutes. This is shorter than the mean half-life of some opiates, necessitating repeated administration when prolonged inhibition of opioid receptor triggering is required. Naloxone is primarily metabolized by the liver; its primary metabolic product is naloxone-3-glucuronide, which is excreted in the urine.

[0007] Naltrexone is metabolized in the liver by the enzyme dihydrodiol dehydrogenase, primarily to 6β-naltrexol. Other metabolic products include 2-hydroxy-3-methoxy-6β-naltrexol and 2-hydroxy-3-methoxy-naltrexone. These intermediates are then conjugated with glucuronides for further metabolism. The plasma half-lives of naltrexone and its metabolite, 6β-naltrexol, are approximately 4 and 13 hours, respectively.

[0008] Randox Toxicology (Crumlin, United Kingdom) offers a naloxone assay based on BioChip Array Technology (BAT), which is based on the principle of enzyme-linked immunosorbent assay (ELISA). Biochip Array Technology is a highly accurate multiplex testing platform that allows for the simultaneous quantitative or qualitative detection of a wide range of analytes from a single sample. The Biochip is a 3D array of immobilized antibodies specific for various drug compounds, This is a solid-state device with individual test sites that are standardized. A competitive chemiluminescent immunoassay is then used, providing highly sensitive screening. However, with this assay, the level of naltrexone cross-reactivity is 12.5% ​​and the level of naloxone 3-BD glucuronide cross-reactivity is 70.6%.

[0009] Immunalysis Corporation (Pomona, CA) offers ELISA naltrexone assays for oral fluid and forensic use as well, which are based on ELISA principles (see, eg, catalog numbers 239-0096 and 239-0480).

[0010] Neogen Corporation (Lansing, MI) offers a Naltrexone / Nalbuphine ELISA kit, 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 numbers 133015 and 133019).

[0011] Other methods for detecting naloxone and naltrexone include high-performance liquid chromatography / tandem mass spectrometry (LC-MS / MS) analysis. For example, NMS Labs (Willow Grove, PA) offers an LC-MS / MS analysis for naltrexone (total) and the metabolite 6-beta-naltrexol (total) in urine (see, e.g., Test Code 3116U, Test Name Naltrexone and Metabolites - Total (Conjugated / Unconjugated), Urine). Summary of the Invention [Problem to be solved by the invention]

[0012] However, there are currently no known homogeneous immunoassays available for either or both of these two drugs. Thus, there is a need in the art for monoclonal antibodies that specifically bind to naloxone and / or naltrexone, and for 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 measurements thereof.

[0013] Furthermore, given the similar structure of naloxone and naltrexone to various opiates (see FIG. 1), these drugs may cross-react in various opiate assays. In particular, it has been commonly observed that various opiate assays show false-positive results for patients receiving naloxone / naltrexone treatment. Therefore, there is also a need in the art to neutralize naloxone and naltrexone present in biological samples, thereby preventing any false-positive or false-high opiate measurements, and thus providing more accurate qualitative screening and quantitative measurements in clinical opiate assays. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the chemical structures of naloxone and naltrexone, as well as various opiates with which naloxone and naltrexone cross-react in various opiate assays. [Figure 2] FIG. 1 is a schematic diagram of the synthesis of naltrexone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates via C-3 attachment. [Figure 3] FIG. 1 is a schematic diagram of the synthesis of naloxone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates via C-3 attachment. [Figure 4] FIG. 1 is a schematic diagram of the synthesis of naltrexone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates via C-6 attachment. [Figure 5] FIG. 1 is a schematic diagram of the synthesis of naloxone hapten, KLH immunogen, and ovalbumin and G6PDH conjugates via C-6 attachment. [Figure 6-1] FIG. 1 is a graph showing ELISA inhibition assays for various opiates performed using various bleeds obtained from selected mice immunized with naloxone or naltrexone-KLH conjugate. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 7-1] FIG. 1 is a graph showing an ELISA inhibition assay for five selected anti-naloxone monoclonal antibodies using eight major opioids (immunogens indicated at the bottom right of the figure). [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 8-1] FIG. 1 is a graph showing an ELISA inhibition assay for eight selected anti-naltrexone monoclonal antibodies using eight major opioids (immunogens shown in the lower panel of the figure). [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 8-4] Continued from Figure 8-3. [Figure 8-5] Continued from Figure 8-4. [Figure 9] 1 is a graph showing a combined naloxone and naltrexone specific assay using monoclonal antibody 179A 6H6 (generated using a naloxone-containing immunogen). [Figure 10] 1 is a graph showing a combined naloxone and naltrexone specific assay using monoclonal antibody 180C 2A12 (generated using a naltrexone-containing immunogen). [Figure 11] FIG. 1 illustrates evaluation of monoclonal antibody 180A 3D3 in an opiate enzyme-amplified immunoassay. [Figure 12] FIG. 1 illustrates evaluation of monoclonal antibody 179A 6H6 in an opiate enzyme-amplified immunoassay. [Figure 13] FIG. 1 illustrates evaluation of monoclonal antibody 180C 2A12 in an opiate enzyme-amplified immunoassay. DETAILED DESCRIPTION OF THE INVENTION

[0015] Before describing at least one embodiment of the present disclosure in detail with illustrative terms and results, it should be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Therefore, the phrases used herein are intended to have the broadest possible scope and meaning; and the embodiments are intended to be illustrative, not exhaustive. Also, it should be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

[0016] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature utilized in connection with, and techniques for, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and the like. Synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, 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)), both incorporated herein by reference. The nomenclature utilized in connection with, and the laboratory procedures and techniques for, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparations, formulations, and delivery, and treatment of patients.

[0017] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0018] All of the compositions, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, kits, and / or methods have been described with reference to 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 in 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 substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0019] As utilized in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0020] The use of the terms "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings 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 dictates otherwise. Thus, for example, reference to a "compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to two or more.

[0021] Use of the term "at least one" will be understood to include one and any amount greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more depending on the term to which it is connected; furthermore, an amount of 100 / 1000 should not be considered limiting, as higher upper limits may also produce satisfactory results. Furthermore, use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Use of ordinal numbers The use of words (i.e., "first," "second," "third," "fourth," etc.) is only to distinguish between two or more items and is not meant to imply, for example, any sequence or order or importance of one item relative to another, or any order of addition.

[0022] Use of the term "or" in the claims is used to mean an inclusive "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: 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 A and B are both true (or exist).

[0023] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, configuration, structure, or feature described in connection with an embodiment is included in at least one embodiment. The appearances of the phrases "in some embodiments" or "one example" in various places in this specification do not necessarily all refer to, for example, the same embodiment. Moreover, any reference to one or more embodiments or examples should not be construed as a limitation on the scope of the claims.

[0024] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists among test subjects. For example, but not by way of limitation, when the term "about" is used, the specified value may vary from the stated value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, as such variations are appropriate for performing the disclosed method and would be understood by one of ordinary skill in the art.

[0025] As used in this specification and claims, the words "comprising" (and any form of including, 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.

[0026] The term "or combinations thereof," as used herein, refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will typically use an item or term in any combination unless otherwise clear from the context. It will be understood that there is no limit to the number of

[0027] 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 significant extent or degree. For example, when relating to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within close proximity to each other but not 100% adjacent to each other, or that a portion of one of two items is within close proximity to the other item but not 100% adjacent to the other item.

[0028] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains the same basic carbon skeleton and carbon functionality in its structure as a given compound, but may additionally contain one or more substitutions. The term "substituted," as used herein, will be understood to refer to the replacement of at least one substituent of a compound with a residue, R. In certain non-limiting embodiments, R is selected from H, hydroxyl, thiol, halide (selected from fluoride, chloride, bromide, or iodide), C1-C4 compounds (selected from one of the following: optionally substituted linear, branched, or cyclic alkyl, and linear, branched, or cyclic alkenyl, where the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl). ), each of which is optionally substituted, the optional substituents being selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, —NH(alkyl), —NH(cycloalkyl), carboxy, and —C(O)-alkyl.

[0029] The term "sample," as used herein, will be understood to include any type of biological sample utilized in accordance with the present disclosure. Examples of fluid biological samples utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural effusion, nasopharyngeal fluid, combinations thereof, and the like.

[0030] The term "specific binding partner," as used herein, will be understood to refer to any molecule that can specifically bind to a macrophilin-binding pharmaceutical for the purpose of detecting it. For example, but not by way of limitation, a specific binding partner may 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 pharmaceutical.

[0031] The term "antibody" is used herein in the broadest sense and includes, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof (including, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single chain antibodies, and fragments of at least one of the variable regions of an intact antibody) that exhibit the desired biological activity of the analyte to which they are bound. The term "antibody" refers to antibodies, including those containing the antibody fragments and other antibody fragments that retain the antibody fragment portion, 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).

[0032] 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 generic term referring to naturally occurring proteins, protein fragments, or analogs of a polypeptide sequence. Thus, naturally occurring proteins, protein fragments, and analogs are species of the polypeptide genus. 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; depending on its origin or derivation, an "isolated peptide / polypeptide / protein" is: (1) not associated with peptides / polypeptides / proteins found in nature; (2) free from other peptides / polypeptides / proteins from the same source, e.g., free from mouse proteins; (3) expressed by cells from a different species; and / or (4) not naturally occurring.

[0033] As used herein, the term "amino acid" includes all molecules, whether natural or synthetic, that contain both amino and acid functionalities and that can be 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.

[0034] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from 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. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation 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 a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, (2) is linked to a polynucleotide with which it is not naturally linked, or (3) does not occur in nature as part of a longer sequence.

[0035] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment is ligated. Another type of vector is a viral vector, in which an additional DNA segment is ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can Upon introduction into a host cell, they can be integrated into the host cell's genome, thereby replicating along with the host genome. Moreover, certain vectors are capable of driving the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0036] The term "naturally occurring" as used herein when applied to an object refers to the fact that the object is found in nature.For example, the polynucleotide or polypeptide sequence that exists in organisms (including viruses) that can be isolated from natural sources and is not intentionally or otherwise modified by humans in the laboratory is naturally occurring.The term "naturally occurring" is used herein interchangeably with the term "native".

[0037] The term "selectively hybridize" as used herein means to detectably and specifically bind. Polynucleotides, oligonucleotides, and fragments thereof encoding peptides / polypeptides / proteins according to the present invention selectively hybridize to nucleic acid strands under hybridization and washing conditions that minimize detectable binding to nonspecific nucleic acids. High stringency conditions are 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 according to the present invention and the nucleic acid sequence of interest will be at least 80%, more typically at least 85%, 90%, 95%, 99%, and 100%, with increasing homology. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum correspondence. Gaps (in either of the two sequences being matched) are allowed in maximum correspondence; a gap length of 5 or less is preferred (but not limited), and 2 or less is more preferred (but not limited). Alternatively, two protein sequences (or polypeptide sequences at least 30 amino acids in length derived therefrom) are homologous (as that term is used herein) if they have an alignment score of more 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 (Vol. 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. Two sequences, or portions thereof, are more preferably homologous if their amino acids are 50% or more identical when optimally aligned using the ALIGN program.The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence (i.e., identical without being 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 a portion of a reference polynucleotide sequence. By way of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0038] The following terms are used to describe 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 a defined sequence used as a basis for sequence comparison; a reference sequence may be a subset of a larger sequence (e.g., as a segment of a full-length cDNA or gene sequence set forth in a sequence listing) or may comprise the complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. The length of a polynucleotide or amino acid sequence is an amino acid length. Because two polynucleotide or amino acid sequences may each contain (1) similar sequences between the two molecules (i.e., a portion of the complete polynucleotide or amino acid sequence), or (2) sequences that differ between the two polynucleotide or amino acid sequences, sequence comparison between two (or more) molecules is typically performed by comparing 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 contiguous nucleotide positions or 6 amino acids, where a polynucleotide or amino acid sequence is compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acids, and the portion of the polynucleotide sequence in the comparison window may contain no more than 20 percent additions, deletions, substitutions, etc. (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) of the optimal alignment of the two sequences. Optimal alignment of sequences to align a comparison window can be performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 2:482 (1981)), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443 (1970)), the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 85:2444 (1988)), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Geneworks, or MacVector software packages in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) produced by the various methods is selected.

[0039] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., nucleotide-by-nucleotide or residue-by-residue) over the comparison window.The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over the 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 both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying this result by 100 to obtain the percentage of sequence identity. The term "substantial identity," as used herein, refers to a characteristic of a polynucleotide or amino acid sequence, including a sequence having at least 85 percent sequence identity, e.g., at least 90-95 percent sequence identity, or at least 99 percent sequence identity, relative to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions totaling no more than 20 percent of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0040] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd ed., E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), α,α-disubstituted amino acids, unnatural amino acids such as N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, α-carboxyglutamic acid, ε-N,N,N-trimethyllysine, Examples include 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 the 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.

[0041] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, e.g., at least 90 percent sequence identity, or at least 95 percent sequence identity, or at least 99 percent sequence identity. In certain (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 having similar side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine ​​and methionine. Particular conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0042] The term "variant" of a reference polypeptide refers to a polypeptide having one or more amino acid substitutions, deletions, or insertions compared to the reference polypeptide. Amino acid substitutions may be "conservative" or "non-conservative." A "conservative" amino acid substitution refers to the substitution 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 that are related in their side chains. Genetically encoded amino acids are generally classified into families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More specifically, the 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, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments or analogs of antibody or immunoglobulin molecules are readily produced by those skilled in the art. Preferred amino and carboxy termini of 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 to public or proprietary sequence databases.In a specific, but non-limiting, embodiment, computer comparison methods are used to identify sequence motifs or predicted protein conformation 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, 25). 3:164 (1991)). Thus, the above examples demonstrate that one of skill in the art can recognize the sequence motifs and structural conformations that are used to define structural and functional domains according to the present disclosure.

[0043] 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 other physicochemical or functional properties of such analogs. Analogs can 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) are made in the naturally occurring sequence (such as, but not limited to, portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt helices occurring in the parent sequence or 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., W.H. 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.

[0044] The term "polypeptide fragment," as used herein, refers to a polypeptide that has an amino- and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence. A polypeptide fragment may be of any length, up to and including the length of the reference polypeptide.

[0045] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Thus, the term "antibody" or "antibody peptide" refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or a binding fragment thereof that competes with the intact antibody for specific antigen binding. Binding fragments are produced by recombinant DNA methods and by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (Nature Med., 9:129-134 (2003)).

[0046] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody is performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic methods and screened for antigen binding in the same manner as intact antibodies.

[0047] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0048] "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0049] The term "CDR," and its plural "CDRs," refers to the complementarity-determining regions (CDRs) of an antibody or antibody fragment, which determine 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). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences comprising scaffolding or framework regions. Among the various CDRs, CDR3 sequences, particularly CDRH3, are the most diverse and therefore contribute most strongly to antibody specificity. There are at least two approaches to determining CDRs: (1) an approach based on cross-species sequence diversity (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) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989)), which is incorporated by reference in its entirety).

[0050] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants typically include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a "conformational epitope") and specific charge characteristics.

[0051] An epitope is defined as "identical" to another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides with different primary amino acid sequences may contain identical epitopes. In certain embodiments, identical epitopes may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope when they compete for specific binding to the same epitope.

[0052] An antibody "specifically binds" an antigen if it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody comprises an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, an antibody can bind different antigens, so 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 may bind to a specific antigen, such as a 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 Specifically binds to an antigen with a dissociation constant of less than or equal to M. When an antibody specifically binds to a receptor or ligand (i.e., a counter-receptor), the antibody can substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when excess antibody reduces the amount of receptor bound to the ligand by at least about 20%, 40%, 60%, or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0053] An "isolated" antibody is one that has been separated and / or recovered from components of the environment in which it is produced. Contaminating components of the production environment are substances that interfere with diagnostic or therapeutic uses and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be measurably purified by at least three different methods: 1) to greater than 50% by weight of the antibody, e.g., greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight, as determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence, e.g., at least 15 residues of sequence, by use of a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or alternatively silver staining. An isolated antibody comprises the antibody in situ within recombinant cells, since at least one component of the environment in which the antibody is produced is absent. However, an isolated antibody will usually be produced by at least one purification step. Furthermore, an "isolated antibody" will be substantially free of other antibodies with different antigenic specificities. However, the isolated antibodies may have some cross-reactivity to other related antigens.

[0054] 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 variants necessarily have less than 100% sequence identity or similarity with an amino acid sequence that has at least 75%, e.g., at least 80%, or at least 85%, or at least 90%, or at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody.

[0055] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies that specifically bind to the same epitope. That is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a single production method, by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, in one embodiment, monoclonal antibodies produced according to the present disclosure can be produced by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0056] Monoclonal antibodies utilized in accordance with the present disclosure can be produced by any methodology known in the art, including, but not limited to, the result of a deliberate immunization protocol; the result of an immune response that naturally leads to the production of antibodies during the course of disease or cancer; phage-derived antibodies, etc. In addition to the hybridoma production methods listed above, monoclonal antibodies of the present disclosure can be produced by a variety of other methods, including, 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)).

[0057] Once antibodies are obtained, for example, individual B cells can be identified and / or monoclonal antibodies can be generated. Once antibodies are produced, sequences encoding the variable regions of these antibodies are obtained. Variable region sequences can be obtained, for example, by first sequencing antibody proteins produced by hybridomas, B cells, or phages to determine the encoding nucleic acid sequence. In one embodiment, immunoglobulin variable region (VH and VL) DNA or cDNA is instead sequenced. If the antibodies are derived from hybridoma cell lines or isolated B cells, cDNA encoding the variable regions can be amplified using PCR, for example, by the methods described in Babcook et al. (Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996)) and PCT Publication No. WO 92 / 02551. The contents of both references are incorporated herein by reference in their entirety.

[0058] The term "neutralizing antibody" or "neutralizing antibody" refers to an antibody that reduces at least one activity of a polypeptide that contains an epitope to which the antibody specifically binds. In certain embodiments, a neutralizing antibody reduces an activity in vitro and / or in vivo.

[0059] The term "antigen-binding site" refers to a portion of an antibody capable of specifically binding an antigen. In certain embodiments, the antigen-binding site is provided by one or more antibody variable regions.

[0060] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, the target species is more abundant than any other individual species in the composition). Generally, a substantially pure composition will contain greater than about 50%, e.g., greater 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 essential homogeneity (no contaminating species are detectable in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0061] The term "agent" refers to a chemical substance, a mixture of chemical substances, a biological polymer, or an extract made from biological materials. In certain embodiments, the "agent" may be a monoclonal antibody according to the present disclosure.

[0062] The term "antagonist" refers to an agent that reduces the activity of a protein / enzyme.

[0063] The term "agonist" refers to an agent that increases the activity of a protein / enzyme.

[0064] The term "carrier protein," as used herein, will be understood to refer to an immunogenic protein that can be attached to small molecules that are typically not immunogenic (such as, but not limited to, drugs, organic compounds, and peptides and oligosaccharides having a molecular weight of less than 2-5 kDa) for the purpose of stimulating an immune system response in the form of producing antibodies against the small molecule.

[0065] Turning now to the inventive concept, 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 being shown below as Formulas I and II, respectively. [ka]

[0066] In certain non-limiting embodiments, 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 producing antibodies against naloxone and / or naltrexone. Non-limiting examples of carrier proteins utilized by the present disclosure include KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), ovalbumin, BTG (bovine thyroglobulin), and BGG (bovine gamma globulin).

[0067] In certain non-limiting embodiments, the carrier protein is attached to a specific carbon position of naloxone and / or naltrexone. For example (and not by way of limitation), the carrier protein is 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 below in Formulas III-VIII. Note that each of these formulas covers both naloxone and naltrexone, and that 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).

[0068] [ka] 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-, lower alkyl (C0-C 10), -CONH-, -SO2-, -PO4-, etc.); C is a carrier protein (for example, but not limited to: KLH, BSA, OVA, BGG, G6PDH, or poly(amino acid)); and R2 is H, alkyl, allyl, or methylenecyclopropyl.

[0069] [ka] In Formula IV: D is a heteroatom (such as, but not limited to, O-, S-, CO-, or CH2-); E is lower alkyl, CO2-, CONH-, SO2-, or PO4-; F is a carrier protein (such as, but not limited to, KLH, BSA, OVA, BGG, G6PDH, or a poly(amino acid)); R2 is H, alkyl, allyl, or methylenecyclopropyl; and R1 is H or CH3.

[0070] [ka] In Formula V: D is a heteroatom (for example, but not limited to, O-, S-, CO-, or CH2-); E is lower alkyl, CO2-, CONH-, SO2-, or PO4-; F is a carrier protein (for example, 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).

[0071] [ka] In Formula VI: G is a linking group containing at least two of the following: a lower alkyl chain, CO2-, CONH-, SO2-, and PO4-; H is a carrier protein (such as, but not limited to, KLH, BSA, OVA, BGG, G6PDH, or poly(amino acids)); R1 is H or CH3; R2 is H, alkyl, aryl, or methyl. It is cyclopropyl.

[0072] [ka] 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 a poly(amino acid)); R1 is H or CH3; and R2 is H, alkyl, allyl, or methylenecyclopropyl.

[0073] [ka] In Formula VII: L is a heteroatom (such as, but not limited to, O, S, CO, or CH); M is a lower alkyl chain, CO-, CONH-, SO-, or PO-; N is a carrier protein (such as, but not limited to, KLH, BSA, OVA, BGG, G6PDH, etc.); R is H or CH; and R is H, alkyl, allyl, or methylenecyclopropyl.

[0074] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof specifically binds to a conjugate comprising OVA attached to the 3-carbon and / or 6-carbon positions of naloxone and / or naltrexone.

[0075] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is allyl), wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO:3; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO:4; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO:6; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO:7; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO:8. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0076] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 22; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 23; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 24; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 26; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 27; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 28. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0077] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 42; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 43; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 44; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 46; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 47; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 48. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0078] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 62; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 63; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 64; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 66; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 67; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 68. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0079] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 82; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 83; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 84; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 86; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 87; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 88. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0080] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, the antibody comprising: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 102; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 103; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 104; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 106; (v) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 107; and (vi) one or more of a light chain variable region CDR3 having the 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) to (vi). In another specific, non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0081] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 122; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 123; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 124; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 126; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 127; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 128. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0082] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 142; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 143; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 144; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 146; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 147; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 148. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0083] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 162; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 163; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 164; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 166; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 167; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 168. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0084] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 182; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 183; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 184; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 186; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 187; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 188. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0085] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 202; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 203; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 204; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 206; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 207; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 208. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0086] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 222; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 223; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 224; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 226; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 227; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 228. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0087] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone, wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 242; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 243; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 244; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 246; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 247; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 248. In certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of the above (i) to (vi).

[0088] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof is at least about 70% identical to any of the heavy chain variable region amino acid sequences outlined in Tables 2-14 (i.e., SEQ ID NOs: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, or 257), including (but not limited to) SEQ ID NOs: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, or 257. 7, 237, or 257.

[0089] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof is selected from the group consisting of SEQ ID NOs: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, or 257 and 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, and have heavy chain variable regions that comprise amino acid sequences that differ by 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 certain (but non-limiting) embodiments, instead of and / or in addition to the above embodiments, the antibody or functional fragment thereof has a light chain variable region amino acid sequence at least about 70% identical to any of the light chain variable region amino acid sequences outlined in Tables 2-14 (i.e., SEQ ID NOs: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, or 258), such as (but not limited to) SEQ ID NOs: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, or 258. 58, 178, 198, 218, 238, or 258.

[0091] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof has a light chain variable region comprising an amino acid sequence that 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.

[0092] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof has a heavy chain variable region comprising an amino acid sequence that 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 functional fragment thereof has a light chain variable region comprising an amino acid sequence that 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 certain (but non-limiting) embodiments, the antibody or functional fragment thereof has a heavy chain variable region comprising an amino acid sequence that differs by fewer than about 12 amino acids from SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, or 257, and / or has a light chain variable region comprising an amino acid sequence that differs by fewer than about 12 amino acids from SEQ ID NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, or 258.

[0093] In another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, or 257, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, or 258.

[0094] In another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has at least about 70% identity to any of the heavy chain amino acid sequences outlined in Tables 2-14 (i.e., SEQ ID NOs: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241), including (but not limited to) SEQ ID NOs: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241. 1, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241.

[0095] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof is selected from the group consisting of SEQ ID NOs: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241 and 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 2 amino acids, less than about 2 and having heavy chains that comprise amino acid sequences that differ by fewer than 3 amino acids, fewer than about 22 amino acids, fewer than about 21 amino acids, fewer than about 20 amino acids, fewer than about 19 amino acids, fewer than about 18 amino acids, fewer than about 17 amino acids, fewer than about 16 amino acids, fewer than about 15 amino acids, fewer than about 14 amino acids, fewer than about 13 amino acids, fewer than about 12 amino acids, fewer than about 11 amino acids, fewer than about 10 amino acids, fewer than about 9 amino acids, fewer than about 8 amino acids, fewer than about 7 amino acids, fewer than about 6 amino acids, fewer than about 5 amino acids, fewer than about 4 amino acids, fewer than about 3 amino acids, fewer than about 2 amino acids, or fewer than about 1 amino acid.

[0096] In certain (but non-limiting) embodiments, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment thereof has at least about 70% identity to any of the light chain amino acid sequences outlined in Tables 2-14 (i.e., SEQ ID NOs: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245), including (but not limited to) SEQ ID NOs: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245. 5, 165, 185, 205, 225, or 245. In some embodiments, the antibody has a light chain comprising an amino acid sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 245.

[0097] In specific (but non-limiting) embodiments, the antibody or functional fragment thereof is selected from the group consisting of SEQ ID NOs: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245 and 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 20 amino acids, less than about 21 amino acids, less than about 2 ... In some embodiments, the light chain comprises an amino acid sequence that differs by fewer than about 18 amino acids, fewer than about 17 amino acids, fewer than about 16 amino acids, fewer than about 15 amino acids, fewer than about 14 amino acids, fewer than about 13 amino acids, fewer than about 12 amino acids, fewer than about 11 amino acids, fewer than about 10 amino acids, fewer than about 9 amino acids, fewer than about 8 amino acids, fewer than about 7 amino acids, fewer than about 6 amino acids, fewer than about 5 amino acids, fewer than about 4 amino acids, fewer than about 3 amino acids, fewer than about 2 amino acids, or fewer than about 1 amino acid.

[0098] In certain (but non-limiting) embodiments, the antibody or functional fragment thereof has a heavy chain comprising an amino acid sequence that 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 functional fragment thereof has a heavy chain comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245. In certain (but non-limiting) embodiments, the antibody or functional fragment thereof has a heavy chain comprising an amino acid sequence that differs by fewer than about 46 amino acids from SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241, and / or a light chain comprising an amino acid sequence that differs by fewer than about 24 amino acids from SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245.

[0099] In another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, or 241, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, or 245.

[0100] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment comprises a heavy chain encoded by a polynucleotide that is at least about 70% identical 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 that is at least about 70% identical to SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, or 259), including (but not limited to) SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249. 9, 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.

[0101] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment comprises a light chain encoded by a polynucleotide at least about 70% identical 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 at least about 70% identical to 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. , 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.

[0102] In further specific (but non-limiting) embodiments, the antibody or functional fragment has a heavy chain encoded by a polynucleotide that is at least about 70% identical to 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 that is at least about 70% identical to SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253. Has.

[0103] In further specific (but non-limiting) embodiments, the antibody or functional fragment has a heavy chain variable region encoded by a polynucleotide that is at least about 70% identical to 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 encoded by a polynucleotide that is at least about 70% identical to SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260.

[0104] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment thereof differs from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249 by a sequence that differs by fewer than about 100 nucleotides, fewer than about 90 nucleotides, fewer than about 80 nucleotides, fewer than about 75 nucleotides, fewer than about 70 nucleotides, fewer than about 60 nucleotides, fewer than about 50 nucleotides, fewer than about 45 nucleotides, fewer than about 40 nucleotides, fewer than about 35 nucleotides, fewer than about 30 nucleotides, fewer than about 25 nucleotides, fewer than about 20 nucleotides, fewer than about 15 nucleotides, fewer than about 10 nucleotides, fewer than about 9 nucleotides, fewer than about 8 nucleotides, fewer than about 7 nucleotides, fewer than about 6 nucleotides, fewer than about 5 nucleotides, fewer than about 4 nucleotides, fewer than about 3 nucleotides, fewer than about 2 nucleotides, or less than about 1 nucleotide. (and / or a heavy chain variable region encoded by a sequence that differs from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, or 259 by 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).

[0105] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, the antibody or functional fragment thereof is selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249 and 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 ... a light chain encoded by a sequence that differs by fewer than about 5 nucleotides, fewer than about 4 nucleotides, fewer than about 3 nucleotides, fewer than about 2 nucleotides, or fewer than about 1 nucleotide (and / or by fewer than about 100 nucleotides, fewer than about 90 nucleotides, fewer than about 80 nucleotides, fewer than about 75 nucleotides, fewer than about 70 nucleotides, fewer than about 60 nucleotides, fewer than about 50 nucleotides, fewer than about 45 nucleotides, fewer than about 40 nucleotides, fewer than about 35 nucleotides, fewer than about 30 nucleotides, fewer than about 25 nucleotides, fewer than about 20 nucleotides, fewer than about 15 nucleotides, fewer than about 10 nucleotides, fewer than about 9 nucleotides, fewer 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).

[0106] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain encoded by a sequence that differs by fewer than about 100 nucleotides from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249, and / or a light chain encoded by a sequence that differs by fewer than about 70 nucleotides from SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253.

[0107] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment thereof has a heavy chain variable region encoded by a sequence that differs by less than about 35 nucleotides from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, or 259, and / or a light chain variable region encoded by a sequence that differs by less than about 34 nucleotides from SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260.

[0108] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0109] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0110] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain (but non-limiting) embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof includes two, three, four, or five of (i) to (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof includes all of (i) to (vi) above.

[0111] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0112] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0113] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0114] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain (but non-limiting) embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof The fragment comprises two, three, four, or five of (i) through (vi) above. In another specific, non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0115] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naltrexone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0116] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0117] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0118] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone (and in certain (but non-limiting) embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof or two, three, four, or five of (i) to (vi) above. In another specific, non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) to (vi) above.

[0119] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i) through (vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i) through (vi) above.

[0120] Certain non-limiting embodiments of the present disclosure relate to an antibody or functional fragment thereof that specifically binds to naloxone (and in certain, but non-limiting, embodiments, a conjugate of Formula III where R2 is cyclopropyl), wherein the antibody comprises one or more of: (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 certain non-limiting embodiments, the antibody or functional fragment thereof comprises two, three, four, or five of (i)-(vi) above. In another specific non-limiting embodiment, the antibody or functional fragment thereof comprises all of (i)-(vi) above.

[0121] The antibody or functional fragment thereof may be a monoclonal antibody or functional fragment thereof, or may be a polyclonal antibody or functional fragment thereof.

[0122] In certain non-limiting embodiments, the antibody or functional fragment thereof is further defined as being selected from a full-length immunoglobulin molecule, an scFv, a Fab fragment, a Fab' fragment, F(ab')2, an Fv, a disulfide-linked Fv, and combinations thereof.

[0123] In certain non-limiting embodiments, the antibody or functional fragment thereof is isolated. In certain (but non-limiting) embodiments, the antibody or functional fragment thereof is purified.

[0124] The present disclosure also relates to antibodies or functional fragments thereof that bind to the same epitope as any of the antibodies or functional fragments described herein above.

[0125] Certain non-limiting embodiments of the present disclosure also relate to methods for producing antibodies or functional fragments thereof capable of specifically binding to naloxone and / or naltrexone, the methods comprising 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.

[0126] Certain non-limiting embodiments of the present disclosure also relate to methods for making antibodies or functional fragments thereof capable of specifically binding to naloxone and / or naltrexone. The methods include binding a carrier protein conjugate to naloxone and / or naltrexone. The method includes immunizing a non-human animal with an antigenic compound containing the conjugate, thereby inducing B cells that produce antibodies that bind to the conjugate; and obtaining the 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) hybridoma technology or B cell PCR technology. The method may further include the optional step of further selecting the antibodies or functional fragments thereof based on their binding to naloxone and / or naltrexone.

[0127] Certain non-limiting embodiments of the present disclosure relate to hybridomas that produce any of the antibodies or functional fragments thereof described herein above.

[0128] Certain non-limiting embodiments of the present disclosure relate to methods for producing an antibody or functional fragment thereof capable of specifically binding to naloxone and / or naltrexone, in which a hybridoma capable of producing any of the antibodies or functional fragments thereof described or otherwise contemplated herein is cultured to produce any of the antibodies or functional fragments thereof described herein. In certain non-limiting embodiments, the antibody or functional fragment thereof is recovered.

[0129] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any of the antibodies or functional fragments thereof disclosed or otherwise contemplated herein attached to a detectable label. Non-limiting examples of detectable labels utilized by the disclosure include enzyme labels, radioactive labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, and particulate labels, and any combination thereof. Furthermore, the detectable label can be attached to the antibody or functional fragment via direct or indirect binding.

[0130] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any of the antibodies or functional fragments thereof disclosed or otherwise contemplated herein attached to a solid support. Attachment of the antibody / functional fragment to the solid support (via direct or indirect conjugation) provides an affinity purification chromatography substrate, such as a column.

[0131] Certain non-limiting embodiments of the present disclosure relate to polynucleotides encoding any of the antibodies or functional fragments thereof disclosed or otherwise contemplated herein.

[0132] In certain (but non-limiting) embodiments, a portion of the polynucleotide encoding the heavy chain of the antibody or functional fragment thereof is at least about 70% identical to 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 functional fragment thereof is at least about 70% identical to 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.

[0133] In yet another specific (but non-limiting) embodiment, and alternatively and / or in addition to the above embodiments, a polynucleotide encoding the light chain of an antibody or functional fragment thereof is A portion of the polynucleotide encoding the light chain variable region of the antibody or functional fragment thereof is at least about 70% identical to 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 functional fragment thereof is at least about 70% identical to SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260), for example (but not limited to) SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253. 3, 213, 233, or 253, and / or SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260.

[0134] In yet another specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain of the antibody or functional fragment thereof is at least about 90% identical to 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 functional fragment thereof is at least about 90% identical to SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253.

[0135] In yet another specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain variable region of the antibody or functional fragment thereof is at least about 90% identical to 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 functional fragment thereof is at least about 90% identical to SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260.

[0136] In another specific (but non-limiting) embodiment, the portion of the sequence corresponding to the heavy chain of the antibody or functional fragment thereof, and / or the portion of the sequence corresponding to the light chain of the antibody or functional fragment thereof, is set forth in SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249, respectively, or SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253 (and / or the portion of the sequence corresponding to the heavy chain variable region of the antibody or functional fragment thereof, and / or the portion of the sequence corresponding to the light chain variable region of the antibody or functional fragment thereof, is set forth in SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, or 259, respectively, or SEQ ID NO: 20, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 8 or 260) by 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.

[0137] In yet another specific (but non-limiting) embodiment, an antibody or functional fragment thereof The portion of the sequence corresponding to the heavy chain differs by less than about 100 nucleotides from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, or 249, and / or the portion of the sequence corresponding to the light chain of the antibody or functional fragment thereof differs by less than about 70 nucleotides from SEQ ID NO: 13, 33, 53, 73, 93, 113, 133, 153, 173, 193, 213, 233, or 253.

[0138] In yet another specific (but non-limiting) embodiment, the portion of the sequence corresponding to the heavy chain variable region of the antibody or functional fragment thereof differs by less than about 35 nucleotides from SEQ ID NO: 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, or 259, and / or the portion of the sequence corresponding to the light chain variable region of the antibody or functional fragment thereof differs by less than about 34 nucleotides from SEQ ID NO: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or 260.

[0139] Certain non-limiting embodiments of the present disclosure relate to vectors comprising any of the polynucleotides encoding the antibodies or functional fragments thereof described or otherwise contemplated herein.

[0140] Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any of the polynucleotides encoding the antibodies or functional fragments thereof described or otherwise contemplated herein. Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any of the vectors described or otherwise contemplated herein.

[0141] Certain non-limiting embodiments of the present disclosure relate to methods for making an antibody or functional fragment thereof capable of specifically binding to naloxone and / or naltrexone, comprising: (a) culturing any of the recombinant host cells described or otherwise contemplated herein in cell culture under conditions that allow for 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.

[0142] Certain non-limiting embodiments of the present disclosure relate to methods for detecting naloxone and / or naltrexone present in a biological sample, comprising: contacting the biological sample with any of the contemplated antibodies or functional fragments thereof disclosed herein or otherwise contemplated under conditions such that antibody / naloxone and / or antibody / naltrexone complexes are formed if naloxone and / or naltrexone, respectively, are present in the sample; and detecting any antibody / naloxone and / or antibody / naltrexone complexes formed, wherein the amount of antibody / naloxone and / or antibody / naltrexone complexes formed is directly proportional to the amount of naloxone and / or naltrexone, respectively, present in the sample.

[0143] In certain (but non-limiting) embodiments, the labels described herein above are attached to antibodies / functional fragments for use in detecting antibody / naloxone and / or antibody / naltrexone complexes.

[0144] Certain non-limiting embodiments of the present disclosure relate to methods for substantially reducing (or substantially eliminating) naloxone and / or naltrexone interference from an opiate assay of a biological sample. The method includes the steps of contacting the biological sample with one or more of the antibodies or functional fragments thereof disclosed or otherwise contemplated herein under conditions such that, if naloxone and / or naltrexone, respectively, are present in the sample, antibody / naloxone and / or antibody / naltrexone complexes are formed; removing any antibody / naloxone and / or antibody / naltrexone complexes formed; and contacting the naloxone with the antibody / naloxone complexes. and performing an opiate assay on the biological sample in which naltrexone and / or naltrexone have been substantially reduced / removed. This method can be utilized with any assay for any opiate and any opiate assay format. For example (but not by way of limitation), the opiate 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 assay, LC / MS / MS, immunoassay, enzyme immunoassay, enzyme-amplified immunoassay, fluorescence polarization immunoassay, combinations thereof, and the like. Various types of opiate assays are well known and commonly used in the art. Therefore, further description thereof is not deemed necessary to enable one skilled in the art to perform the method disclosed herein.

[0145] In certain (but non-limiting) embodiments, the at least one antibody or functional fragment thereof used in the method of substantially reducing (or substantially eliminating) naloxone and / or naltrexone interference from an opiate assay includes 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.

[0146] Certain non-limiting embodiments of the present disclosure also include kits containing any of the antibodies / functional fragments and / or compositions (such as compositions comprising an antibody / functional fragment attached to a detectable label or solid support) disclosed or otherwise contemplated herein, as well as any other reagents utilized in the assays / methods described herein. For example (and not by way of limitation), the kit may further include one or more components of the opiate assays described herein above. For example (and not by way of limitation), the kit may include an antibody or functional fragment thereof that binds to any of the opiates described or otherwise contemplated herein for use in an opiate immunoassay.

[0147] The assay components / reagents of the compositions / kits / methods are provided in any form that enables them to function according to the present disclosure. For example, but not by way of limitation, each of the reagents is provided in liquid form and disposed in the kit in bulk and / or single aliquot form. Alternatively, in certain (but non-limiting) embodiments, one or more of the reagents are disposed in the kit in the form of a single aliquot lyophilized reagent. The use of dried reagents in microfluidic devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0148] In addition to the assay components / reagents detailed herein above, the kit may further contain other reagents for carrying out any of the specific assays described or otherwise contemplated herein. The nature of these additional reagents will depend on the specific assay format, and their identification is well within the skill of those skilled in the art; therefore, further description thereof is deemed unnecessary. Additionally, the components / reagents present in the kit may each be contained in separate containers / compartments, or, depending on the cross-reactivity and stability of the components / reagents, various components / reagents may be combined in one or more containers / compartments. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0149] The relative amounts of the various components / reagents in the kit may be varied widely to provide for concentrations of the components / reagents that substantially optimize the reactions required to occur during the assay method, and also to substantially optimize the sensitivity of the assay. Under appropriate conditions, one or more of the components / reagents in the kit may be provided as a dry powder, such as a lyophilized powder, and the kit may be The kit may further include an excipient for dissolving the dried reagents; in this manner, a reagent solution having an appropriate concentration for performing a method or assay according to the present disclosure can be obtained from these components. Positive and / or negative controls can also be included with the kit. Furthermore, the kit may further include a set of written instructions that explain how to use the kit. Kits of this nature can be used in any of the methods described or otherwise contemplated herein. [Example]

[0150] Examples are provided below. However, it should be understood that the present disclosure should not be limited in its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, the examples are simply provided as one of various embodiments and are intended to be illustrative and not exhaustive. [Example]

[0151] Preparation of immunogens containing naloxone and naltrexone conjugates This example illustrates the preparation of various conjugates containing naloxone or naltrexone through multiple C-position bonds.

[0152] 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 received. 1 H NMR was recorded on a Bruker ULTRASHIELD™ 600 MHz spectrometer (Bruker, Billerica, MA). Chemical shifts were reported in parts per million (ppm, δ) relative to tetramethylsilane as an internal standard or using deuterated solvents. NMR abbreviations used are: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets), ddd (doublet of doublet of doublets), J (coupling constant), Hz (hertz). ESI-MS spectra were recorded on an Agilent HPLC 1290 Single Quad system equipped with a 6130 mass detector at Siemens Healthineers Analytical Facility (Newark, DE). Carry 60 was used for UV:OD280 and BCA concentration measurements.

[0153] The following abbreviations have the meanings set forth below: BCA Assay - Bicinchoninic Acid Assay eq. - equivalent amount g - grams mg - milligram mmol, mM - millimole nm - nanometer CV - column volume cm 2 - square centimeters DCM - dichloromethane MeOH - methanol MeOD-d4 - Deuterated methanol with four deuterium atoms (for NMR spectroscopy) DMF - N,N-dimethylformamide SuOH - N-hydroxysuccinimide NaOAc - sodium acetate AcOH - acetic acid AcO - - Acetate EDAC·HCl - N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide Hydrochloride TFA - Trifluoroacetic acid TLC - Thin Layer Chromatography Rf - retention factor in TLC analysis UV - ultraviolet light v / v - capacity ratio ESI-MS - Electrospray Ionization Mass Spectrometry m / z - mass to charge ratio NMR - nuclear magnetic resonance MHz - Megahertz nG6PDH - native glucose-6-phosphate dehydrogenase enzyme β-NADH - nicotinamide adenine dinucleotide G6PDNa2 - glucose-6-phosphate disodium salt OVA - ovalbumin isolated from chicken egg white mcKLH - Mariculture Keyhole Limpet Hemocyanin (ThermoFisher Scientific) TRIS - Tris(hydroxymethyl)aminomethane KU-nG6PDH dimeric protein complex 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

[0154] FIG. 2 is a schematic diagram of the synthesis of the naltrexone hapten, KLH immunogen, and G6PDH conjugate via C-3 attachment, as described in further detail herein below.

[0155] 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, followed by DMF (5 mL), acetone (5 mL), and potassium carbonate (575 mg, 4.16 mmol, 4.78 equiv.). The resulting reaction mixture was stirred under a nitrogen stream at room temperature (rt.) for 5 min; then, ethyl-5-bromovalerate (375 μL, 495 mg, To the mixture was added 2.36 mmol, approximately 2.71 equivalents. 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 under stirring, and TLC [(DCM / MeOH 8 / 2 v / v) Rf (1) =0.36, Rf (2)=0.80] after 4 h. The solvent was removed on a rotary evaporator to give a white precipitate, which was suspended in acetonitrile (2 × 10 mL) and filtered. The organic extract was concentrated on a rotary evaporator and further dried overnight (16 h) on an oil pump to remove traces of volatiles. The product was suspended in acetonitrile / water (30 / 70 v / v containing 0.1% acetic acid) and injected onto a Shimadzu HPLC system for purification. Fractions containing the desired product were pooled, concentrated on a rotary evaporator, and lyophilized overnight (16–20 h) to give 474.8 mg of compound (2) as a colorless powder in 93% yield. Compound (2): ESI-MS m / z calcd. for: [C 27 H 36 NO6] + 470.3, found [M+H] + = 470.3; 1 H NMR (600 MHz, MeOD-d4) 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, 3.00 Hz, 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).

[0156] Preparation of Compound (3): Compound (2) (474.8 mg, 0.81 mmol) was dissolved in MeOH (5 mL); then, 10 N sodium hydroxide solution (0.53 mL) was added, and the resulting reaction mixture was stirred at room temperature for 2 hours. Completion of the reaction was confirmed by TLC [(DCM / MeOH 8 / 2 v / v), Rf (2) =0.80 Rf (3) =0.20]. The reaction mixture was acidified with HCl (12N, 0.5 mL) in an ice bath. The solvent was removed on a rotary evaporator. The resulting white cake was suspended in acetonitrile (2 mL) and filtered. The filtrate was concentrated on a rotary evaporator. Water containing 0.1% TFA was then added, and the resulting sample was lyophilized overnight (16-20 hours). After lyophilization, 471 mg of the title compound was obtained as a colorless powder in 100% yield. Compound (3): ESI-MS m / z calcd. for: [C 25 H 32 NO6] + 442.2, found [M+H] + = 442.2; 1 H NMR (600 MHz, MeOD-d4) 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).

[0157] 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 equiv.) was added. The resulting reaction mixture was stirred until homogeneous, and then EDAC·HCl (7.30 mg, 0.038 mmol, 2 equiv.) was added. The resulting reaction mixture was stirred at room temperature for 20 hours, and TLC [(DCM / MeOH 8 / 2 v / v), Rf (3) =0.80, Rf (4) =0.20)]. The formation of compound (4) was confirmed by TLC and MS direct loop: [C 29 H 35 N2O8] + Calculated value for ESI m / z=539.2, observed value [M+H] + = 539.3 and [M+Na] + =561.3. This compound (4) was used in the next reaction without further purification.

[0158] Preparation of naltrexone OVA (5): OVA, (12 mg, 2.81 x 10-4 (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 equivalents) was added dropwise to the protein solution under gentle swirling. The resulting reaction mixture was shaken at 4 °C for 20 hours (overnight), and then loaded onto a G-25M SEPHADEX column (GE Healthcare, Chicago, IL) (CV = 70 cm). 3 The conjugate was purified with 2 CV of 50 mM phosphate buffer (pre-equilibrated with 2 CV of 50 mM phosphate buffer, pH 7.00). 280 The elution was monitored at OD. A sharp peak containing the desired naltrexone-OVA conjugate eluted at intervals of 67-90 mL. Naltrexone-OVA (5) was collected and concentrated. The concentration of the conjugate was monitored at OD. 280 The concentration was determined to be 0.89 mg / ml by HPLC. A total of 9 mg of OVA conjugate (5) was obtained in 75% yield.

[0159] 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, Compound (4)-DMF solution (0.488 mL, 7 x 10 -3 20 mg of mcKLH treated with 1000 mM HCl (1 mmol) was used. After purification, the concentration of the conjugate was determined to be 0.96 mg / ml by BCA assay. 15.35 mg of KLH immunogen (6) was obtained in 76.8% yield.

[0160] Preparation of native G6PDH for bioconjugation: Native G6PDH enzyme emulsion (5.5 mL, 5.5 KU, 64.35 mg) was centrifuged at 18,000 RPM at 4°C for 30 minutes. The resulting clear aqueous layer was discarded, and the remaining white cake was suspended in 55 mM TRIS buffer, pH 8.00 (2.00 mL). The suspension was gently swirled until dissolved; it was then loaded onto a G-25M Sephadex column (CV = 196 mL) pre-equilibrated with 55 mM TRIS buffer, pH 8.00. The enzyme was eluted in 75-116 mL intervals. The enzyme concentration was adjusted to 5 mg / mL using an Amicon stirred-cell concentrator (30,000 MW cutoff) and then cooled to 0-4°C using an ice bath. 240 mg of glucose-6-phosphate disodium salt [G6PDNa2] was then added. The resulting reaction mixture was gently swirled for 1 min; then, β-NADH (120 mg) was added, and the resulting reaction mixture was stirred again for 1 min. The enzyme solution was aliquoted into three plastic tubes (1.90 mL per tube, equivalent to 9.50 mg of enzyme). Tubes 1–3, containing native G6PDH, were stored in an ice bath in preparation for the addition of compound (4).

[0161] 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, approximately 3 equiv.) was added. The resulting reaction mixture was stirred until homogeneous, and then EDAC·HCl (7.30 mg, 0.038 mmol, 2 equiv.) was added. The resulting reaction mixture was stirred at room temperature for 22 h and analyzed by TLC [(DCM / MeOH 8 / 2 v / v), Rf (4) =0.80, Rf (3) = 0.20]. After the formation of compound (4), degassed DMF (0.242 μL) was added to prepare a 20 mg / mL compound (4)-DMF solution. [C 29 H 35 N2O8] + Calculated value for ESI m / z=539.3, observed value [M+H]+ =539.3, [M+Na] + =561.3; this compound (4) was used for bioconjugation without further purification.

[0162] Preparation of naltrexone-nG6PDH enzyme (7a-c): To tube 1, compound (4) (243 μL in DMF) was added [10× molar excess of compound ( 4)]. The reaction mixture was rocked at 2-8°C for 1 hour; then, it was quenched with L-lysine [9 μL of a 1 M solution, 10× molar excess of L-lysine relative to each compound (4)]. The reaction mixture was then rocked at room temperature for 20 minutes. The conjugate was dissolved in 55 mM TRIS buffer. A G-50M SEPHADEX® column (CV = 67 cm) pre-equilibrated at pH 7.00 (2 CV) was used. 3 The conjugate was purified by OD ). The conjugate eluted as a sharp peak at intervals 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 by HPLC. 7.93 mg of G6PDH conjugate (7a) was obtained in 83% yield.

[0163] To tube 2, compound 4 (485 μL in DMF) was added (20× molar excess of compound 4 relative to nG6PDH); the reaction mixture was rocked at 2-8°C for 1 hour; then, it was quenched with L-lysine (18 μL of a 1 M solution, 10× molar excess of L-lysine relative to compound 4). The reaction mixture was then rocked at room temperature for 20 minutes. This conjugate was then added to G-50M HCl (pre-equilibrated with 55 mM TRIS buffer, pH 7.00 (2 CV)). SEPHADEX® column (CV = 67 cm 3 The conjugate was purified by OD ). The conjugate eluted as a sharp peak at intervals of 45-60 mL. All fractions containing the desired conjugate were collected and concentrated. The concentration of the conjugate was determined by OD . 280The concentration of G6PDH conjugate (7b) was determined to be 1.25 mg / mL by HPLC. 8.09 mg of G6PDH conjugate (7b) was obtained in 85% yield.

[0164] To tube 3, compound 4 (729 μL in DMF) was added [30× molar excess of compound 4 relative to nG6PDH]. The reaction mixture was rocked at 2-8°C for 1 hour; then, it was quenched with L-lysine [27 μL of a 1 M solution, 10× molar excess of L-lysine relative to compound 4]. The reaction mixture was then rocked at room temperature for 20 minutes. This conjugate was added to G-50M HCl pre-equilibrated with 55 mM TRIS buffer, pH 7.00 (2 CV). SEPHADEX® column (CV = 67 cm 3 The conjugate was purified by OD ). The conjugate eluted as a sharp peak at intervals 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 by HPLC. 8.27 mg of G6PDH conjugate (7c) was obtained in 87% yield.

[0165] FIG. 3 is a schematic diagram of the synthesis of the naloxone hapten, KLH immunogen, and G6PDH conjugate via C-3 attachment, as described in more detail herein below.

[0166] Preparation of Compound (9): This compound was prepared using the same synthetic method as described for the preparation of Compound (2). After lyophilization, Compound (9) (465.5 mg) was obtained as a colorless powder in 99% yield. Compound (9): ESI-MS m / z calcd. for: [C 26 H 34 NO6] + 456.2, found [M+H] + = 456.2; 1H NMR (600 MHz, MeOD-d4) 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).

[0167] Preparation of Compound (10): This compound was prepared using the same synthetic method as described for the preparation of Compound (3). After lyophilization, Compound (10) (447 mg) was obtained as a colorless powder in almost 100% yield. Compound (10): ESI-MS m / z calcd. for: [C 24 H 30 NO6] + = 428.2, found [M+H] + = 428.2; 1 H NMR (600 MHz, MeOD-d4) δ: 6.82 (d, J = 8.22 Hz, 1 H), 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, 5.10Hz, 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).

[0168] Preparation of Compound (11): This compound was prepared using the same synthetic method as described for the preparation of Compound (4).

[0169] Preparation of naloxone OVA (12): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0170] Preparation of Naloxone KLH Immunogen (13): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0171] Preparation of naloxone nG6PDH [14(a-c)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDHs [7(a-c)].

[0172] FIG. 4 is a schematic diagram of the synthesis of the naltrexone hapten, KLH immunogen, and G6PDH conjugate via C-6 attachment, as described in detail herein below.

[0173] 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 overnight (20 hours) at room temperature. TLC analysis of the reaction mixture indicated the completion of the reaction [(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. Water / acetonitrile (1.4 mL / 0.6 mL v / v) containing 0.1% AcOH was then added. The crude reaction mixture was then injected onto a preparative Shimadzu HPLC system for purification. Fractions containing the desired product were pooled together, concentrated on a rotary evaporator, and lyophilized overnight (16-20 hours). This afforded compound (15) (124 mg) as a colorless powder in 75% yield. Compound (15): ESI-MS m / z calcd. for: [C 22 H 27 NO6] + 415.2, found [M+H] + = 415.2, and [M+Na] + = 429.2; 1H NMR (600 MHz, MeOD-d4) 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, 4.04 Hz, 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).

[0174] Preparation of Compound (16): This compound was prepared using the same synthetic method as described for the preparation of Compound (4).

[0175] Preparation of Naltrexone OVA (17): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0176] Preparation of Naltrexone KLH Immunogen (18): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0177] Preparation of naltrexone-nG6PDH [19(a-c)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDH [7(a-c)].

[0178] FIG. 5 is a schematic diagram of the synthesis of the naloxone hapten, KLH immunogen, and G6PDH conjugate via C-6 attachment, as described in detail herein below.

[0179] 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) was added. The resulting reaction mixture was stirred at room temperature overnight. TLC analysis showed 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. Fractions containing the desired product were pooled together, 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 calcd. for: [C 21 H 25 NO6] + 401.2, found [M+H] + = 401.2, [M+Na] + = 423.2; 1H NMR (600 MHz, MeOD-d4) 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).

[0180] Preparation of Compound (21): This compound was prepared using the same synthetic method as described for the preparation of Compound (4).

[0181] Preparation of naloxone OVA (22): This conjugate was prepared using the same synthetic method as described for the preparation of OVA conjugate (5).

[0182] Preparation of Naloxone KLH Immunogen (23): This immunogen was prepared using the same synthetic method as described for the preparation of KLH immunogen (6).

[0183] Preparation of naloxone nG6PDH [24(a-c)]: These nG6PDH conjugates were prepared using the same synthetic method as described for the preparation of nG6PDH [7(a-c)]. [Example]

[0184] Production of anti-naloxone and anti-naltrexone monoclonal antibodies This example demonstrates the use of specific monoclonal antibodies against naloxone and naltrexone. The production of these monoclonal antibodies is described. These monoclonal antibodies can be used in naloxone- and naltrexone-specific assays, respectively.

[0185] Furthermore, these monoclonal antibodies can be used as blocker antibodies in opiate assays (such as, but not limited to, hydrocodone assays). It is commonly observed that various opiate assays exhibit false-positive results for patients receiving naloxone / naltrexone treatment. In such cases, the naloxone / naltrexone-specific assays disclosed herein can be used to screen patients to rule out 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, thus preventing or substantially reducing any naloxone / naltrexone interference observed in quantitative clinical opiate assays.

[0186] In this example, various immunogens containing naloxone or naltrexone attached to KLH at various positions were prepared and used as immunogens to intraperitoneally immunize mice (e.g., BALB / c mice, Swiss Webster mice, or AJ strain mice). Mice were immunized three or more times to generate a high-titer immune response. The primary immunization was performed using an adjuvant such as CFA (immunogen emulsified in CFA), followed by subsequent booster immunizations using an adjuvant such as IFA. After immunization, mice were bled, and serum samples were isolated using centrifugation. Serum samples from these mice were tested for anti-naloxone and anti-naltrexone antibodies using conjugates of naloxone and / or naltrexone with ovalbumin (ovalbumin conjugates). Antibodies were examined for binding to the ovalbumin conjugates and then to the free drug molecules using a microtiter plate ELISA and an inhibition ELISA. Figure 6 shows the inhibition ELISA results using eight key opioid drugs. The immunogens used to generate the mouse bleeds shown in Figure 6 are as follows: Top row, from left: naltrexone 3-KLH (Formula III, where R2 is cyclopropyl); naltrexone 6-KLH (Formula VI, where R2 is cyclopropyl); naloxone 3-KLH (Formula III, where R2 is allyl); and naloxone 3-KLH. Bottom row, from left to right: naltrexone 3-KLH; naltrexone 6-KLH; naloxone 6-KLH (Formula VI, where R2 is allyl); and naloxone 6-KLH.

[0187] Mice with favorable anti-drug antibody titers and binding to free drug in ELISA inhibition assays were selected for further monoclonal antibody generation using a method similar to that described in U.S. Patent No. 9,815,907 (issued November 14, 2017 to Sharma et al.). Specifically, mice with the highest titers and best specificity were boosted 3 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. Approximately 10 days later, 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 expanded and subcloned, and the supernatants were purified using a Protein A SEPHAROSE® (GE Healthcare, Chicago, IL) column. Purified antibody samples were tested for binding to the immunogen (i.e., drug-carrier protein conjugate) and free opioid using ELISA.

[0188] Table 1 lists the names for some of the selected hybridoma clones generated against the naloxone and naltrexone drug molecules described herein.

[0189] [Table 1]

[0190] Antibody solutions (immune mouse bleeds, hybridoma clone supernatants, or purified antibodies) were screened using an inhibition ELISA according to the following protocol. Plates were coated with 50 μL per well of 1 μg / mL naloxone 3-ovalbumin, naloxone 6-ovalbumin, naltrexone 3-ovalbumin, or naltrexone 6-ovalbumin in phosphate-buffered saline. Plate coating was performed at room temperature for at least 1 hour or overnight at approximately 2°C to 8°C. The plates were then gently tapped dry and blocked with 200 μL per well of blocking buffer (0.5% casein solution in PBS containing 0.05% TWEEN® 20 surfactant (Croda International PLC, Snaith, United Kingdom)). Plate blocking was performed by incubating the plate at room temperature for at least 30 minutes with shaking. The plate was washed using a plate washer (BioTek®, Winooski, VT) equipped with a plate stacker, where the wash buffer was MILLIQ® water (Millipore Corporation, Billerica, MA) containing 0.05% TWEEN® 20. The monoclonal antibody to be screened was then added to each well along with the free drug as follows: 25 μL per well of appropriately diluted culture supernatant (or antibody solution) and an additional 25 μL of 20 μg / mL of free drug. Incubation was carried out at room temperature with plate shaking for approximately 1 hour. The plate was washed. Enzyme conjugate (goat anti-mouse IgG conjugated to HRP diluted 1:3000 in blocking buffer) was added at 50 μL per well. Incubation was carried out at room temperature with shaking for approximately 1 hour. The plate was then washed, and a color development solution (TMB from Moss Substrates, Pasadena, MD) was added at a volume of 100 μL per well. If the desired antibody was present in the hybridoma supernatant, a decrease in optical density was observed compared to wells containing free drug. The plate was read at 650 nm wavelength using an ELISA plate reader (Molecular Devices, San Jose, CA).NOTE: The appropriate dilution of antibody solution was determined by performing a titration ELISA where an optical density of approximately 1.0 was observed. If so, antibody dilution was selected.

[0191] As shown in Figure 7, monoclonal antibodies designated 179B 9G1.1, 179B 9E12, 179B 10A7, 179A 6H6, and 179B 9E7 were generated using naloxone-3-Val-mcKLH (Formula III where R2 is allyl) and had good specificity for naloxone compared to eight other major opioids tested in the inhibition ELISA assay. As shown in Figure 8, monoclonal antibodies designated 180B 13F7, 180C 1F8, 180C 7G8, 180C 8A10, 180C 9D5, 180C 9G8, 180C 2A12, and 180A 3D3 were generated using naltrexone-3-Val-mcKLH (Formula III where R2 is cyclopropyl) and had good specificity for naltrexone relative to eight other major opioids tested in the inhibition ELISA assay. Based on these results, the monoclonal antibodies were then used in naloxone / naltrexone-specific immunoassays and in methods to prevent or substantially reduce naloxone / naltrexone interference in other opiate assays, as described in more detail below. [Example]

[0192] Monoclonal antibody sequencing Hybridoma cells prepared as in Example 2 were used to sequence the heavy and light chains of various monoclonal antibodies. The sequences of the various clones were aligned to obtain a consensus sequence. The various sequences obtained for each monoclonal antibody are shown in Tables 2-14 below.

[0193] [Table 2]

[0194] [Table 3]

[0195] Table 4

[0196] Table 5

[0197] Table 6

[0198] Table 7

[0199] Table 8

[0200] Table 9

[0201] Table 10

[0202] Table 11

[0203] Table 12

[0204] Table 13

[0205] [Table 14] [Example]

[0206] Use of monoclonal antibodies in naloxone- and naltrexone-specific assays In this example, the monoclonal antibodies generated in Example 2 were utilized in naloxone- and naltrexone-specific immunoassays to demonstrate the ability of these monoclonal antibodies to detect one or both drugs in a clinical immunoassay.

[0207] Figure 9 is a graphical representation of a naloxone and naltrexone specific assay using anti-naloxone monoclonal antibody 179A 6H6. As can be seen, anti-naloxone The anti-naloxone monoclonal antibodies detect both naloxone and naltrexone with essentially the same specificity. Similar results were obtained with the anti-naloxone monoclonal antibodies 179B 10A7, 179B 9G1, 179B 9E12, and 179B 9E7. Therefore, these antibodies could be used in clinical detection immunoassays for either or both drugs.

[0208] Figure 10 is a graphical representation of a naloxone and naltrexone-specific assay using anti-naltrexone monoclonal antibody 180C 2A12. This anti-naltrexone monoclonal antibody was also able to detect naloxone in addition to naltrexone, albeit with lower specificity compared to naltrexone. However, this antibody could still be used in clinical detection immunoassays for either or both drugs.

[0209] Based on these results, these antibodies can be utilized in the following assay configuration: The EMIT® II (Enzyme-Amplified Immunosorbent Assay, Siemens Healthineers, Newark, DE) plus naloxone and / or naltrexone assay is a homogeneous enzyme immunoassay used for the analysis of 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). Enzyme activity decreases upon antibody binding, 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 an absorbance change measured spectrophotometrically. Endogenous serum G6PDH does not interfere. This is because the coenzyme NAD functions exclusively with the bacterial (Leuconostoc mesenteroides) enzyme used in the assay.

[0210] The EMIT® assay format includes an antibody / substrate reagent known as Reagent 1. This reagent contains the antibody as well as assay buffers, preservatives, and stabilizers. The EMIT® assay format also includes an enzyme reagent known as Reagent 2. This reagent contains the conjugate as well as assay buffers, preservatives, and stabilizers. [Example]

[0211] Reducing naloxone / naltrexone interference in opioid assays In this example, the monoclonal antibodies generated in Example 2 were utilized in opiate enzyme-amplified immunoassays to demonstrate their ability to reduce naloxone / naltrexone interference in these assays.

[0212] Figure 11 demonstrates that in the presence of naltrexone antibody blocker 3D3, naloxone and naltrexone cross-reactivity shifts from a positive to a negative response up to the cutoff (300 ng / mL). Similarly, Figure 12 demonstrates that in the presence of naloxone antibody blocker 6H6, naloxone cross-reactivity shifts from a positive to a negative response up to the cutoff (300 ng / mL) only at much lower concentrations.

[0213] In Figure 13, anti-naltrexone monoclonal antibody 2A12 was used as a blocker in a hydrocodone assay with increasing amounts of naloxone and naltrexone that produced a negative response up to the cutoff (300 ng / ml).

[0214] Thus, there have been provided in accordance with the present disclosure compositions and methods of making and using the same that fully satisfy the objects and advantages set forth above. While the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, many alternatives, modifications, and variations thereon may be realized. It is evident that this is apparent to those skilled in the art and therefore it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. An antibody or functional fragment thereof capable of specifically binding to naloxone and naltrexone, the antibody or functional fragment thereof comprising: (1) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; (2) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 42, 43, and 44, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively; (3) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 82, 83, and 84, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 86, 87, and 88, respectively; (4) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 102, 103, and 104, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 106, 107, and 108, respectively; (5) Heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 146, 147, and 148, respectively; (6) Heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 162, 163, and 164, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 166, 167, and 168, respectively; (7) Heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 182, 183, and 184, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 186, 187, and 188, respectively; (8) Heavy chain variable domains having the amino acid sequences of SEQ ID NOs: 202, 203, and 204, respectively. light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 206, 207, and 208, respectively; (9) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 222, 223, and 224, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 226, 227, and 228, respectively; or (10) The antibody or functional fragment thereof, comprising heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 242, 243, and 244, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 246, 247, and 248, respectively.

2. The antibody or functional fragment thereof of claim 1, further defined as a monoclonal antibody or functional fragment thereof.

3. (1) the heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 17, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 18; (2) the heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 57, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 58; (3) the heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 97, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 98; (4) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 117, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 118; (5) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 157, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 158; (6) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 177, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 178; (7) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 197, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 198; (8) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 217, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 218; (9) The heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 237, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 238; or (10) The antibody or functional fragment thereof according to claim 1, wherein the heavy chain variable region of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO: 257, and / or the light chain variable region of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO:

258.

4. (1) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 17 and 18, respectively; (2) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 57 and 58, respectively; (3) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 97 and 98, respectively; (4) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 117 and 118, respectively; (5) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 157 and 158, respectively; (6) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 177 and 178, respectively; (7) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 197 and 198, respectively; (8) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 217 and 218, respectively; (9) The heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 237 and 238, respectively; or (10) The antibody or functional fragment thereof according to claim 1, wherein the heavy and light chain variable regions of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 257 and 258, respectively.

5. (1) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO: 5; (2) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 41, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 45; (3) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 81, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 85; (4) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 101, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 105; (5) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 141, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 145; (6) the heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 161, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 165; (7) The heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 181, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 185; (8) The heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 201, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 205; (9) The heavy chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 221, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence at least 90% identical to SEQ ID NO: 225; or (10) The antibody or functional fragment thereof according to claim 1, wherein the heavy chain of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO: 241, and / or the light chain of the antibody or functional fragment thereof has an amino acid sequence that is at least 90% identical to SEQ ID NO:

245.

6. (1) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 1 and 5, respectively; (2) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 41 and 45, respectively; (3) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 81 and 85, respectively; (4) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 101 and 105, respectively; (5) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 141 and 145, respectively; (6) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 161 and 165, respectively; (7) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 181 and 185, respectively; (8) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 201 and 205, respectively; (9) The heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 221 and 225, respectively; or (10) The antibody or functional fragment thereof according to claim 1, wherein the heavy and light chains of the antibody or functional fragment thereof have the amino acid sequences of SEQ ID NOs: 241 and 245, respectively.

7. The antibody or functional fragment thereof of claim 1, further defined as being selected from a full-length immunoglobulin molecule, an scFv, a Fab fragment, a Fab' fragment, F(ab')2, an Fv, a disulfide-linked Fv, and combinations thereof.

8. (1) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:9, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:13; (2) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:49, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:53; (3) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:89, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO:93; (4) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 109, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 113; (5) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 149, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 153; (6) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 169, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 173; (7) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 189, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 193; (8) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 209, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 213; (9) the heavy chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 229, and / or the light chain is encoded by a polynucleotide sequence at least 90% identical to SEQ ID NO: 233; or (10) The antibody or functional fragment of claim 1, wherein the heavy chain is encoded by a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 249, and / or the light chain is encoded by a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

253.

9. The antibody or functional fragment thereof of claim 1, further defined as a purified antibody or functional fragment thereof.

10. 1. A composition comprising: At least one antibody or functional fragment thereof according to any one of claims 1 to 9; a detectable label attached to said at least one antibody or functional fragment thereof; The composition comprising:

11. 1. A composition comprising: a solid support; At least one antibody or functional fragment thereof according to any one of claims 1 to 9 bound to said solid support; The composition comprising:

12. A polynucleotide encoding the antibody or functional fragment thereof according to any one of claims 1 to 9.

13. A vector comprising a polynucleotide encoding the antibody or functional fragment thereof according to any one of claims 1 to 9.

14. A recombinant host cell comprising a polynucleotide encoding the antibody or functional fragment thereof according to any one of claims 1 to 9.

15. 1. A method for producing an antibody or functional fragment thereof capable of specifically binding to at least naloxone and naltrexone, comprising: (a) culturing the recombinant host cell of claim 14 in cell culture under conditions that allow expression of the antibody or functional fragment thereof encoded by the polynucleotide; (b) isolating the antibody or functional fragment thereof from the cell culture; The method comprising:

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