title: A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.
abstract: The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not bind or poorly bound to heroin, 6-AM, and morphine. This simple and nonradioactive method can be extended to other platforms, such as oxycodone, cocaine, nicotine, and methamphetamine for the selection of the lead hapten design during substance abuse vaccine development.
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.|U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, 20817, USA.
authors.forename: Oscar B
authors.initials: OB
authors.lastname: Torres
authors.affiliation: Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 9800 Medical Drive, Bethesda, MD, 20892, USA.|National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 9800 Medical Drive, Bethesda, MD, 20892, USA.
authors.forename: Joshua F G
authors.initials: JF
authors.lastname: Antoline
authors.affiliation: Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 9800 Medical Drive, Bethesda, MD, 20892, USA.|National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 9800 Medical Drive, Bethesda, MD, 20892, USA.|WuXi AppTec (Shanghai) Co., Ltd, 288 FuTe Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
authors.forename: Fuying
authors.initials: F
authors.lastname: Li
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.|U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, 20817, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Jalah
authors.affiliation: Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 9800 Medical Drive, Bethesda, MD, 20892, USA.|National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 9800 Medical Drive, Bethesda, MD, 20892, USA.
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Jacobson
authors.affiliation: Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 9800 Medical Drive, Bethesda, MD, 20892, USA.|National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 9800 Medical Drive, Bethesda, MD, 20892, USA.
authors.forename: Kenner C
authors.initials: KC
authors.lastname: Rice
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.
authors.forename: Carl R
authors.initials: CR
authors.lastname: Alving
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA. gmatyas@hivresearch.org.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Matyas
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D000915:Antibody Affinity; D060326:Chemistry Techniques, Synthetic; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003903:Deuterium; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006241:Haptens / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D009020:Morphine / Q000097:blood* / Q000276:immunology; D009022:Morphine Derivatives / Q000097:blood* / Q000276:immunology; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D006241:Haptens; D009022:Morphine Derivatives; D009020:Morphine; D003903:Deuterium; C026979:6-O-monoacetylmorphine
keywords: Antibody affinity; Apparent dissociation constant (K d); Competition ELISA; Equilibrium dialysis; Heroin hapten; UPLC/MS/MS