A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids

Abstract

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide—a useful analog for the study of heroin hapten–antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstractStrategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1060-4) contains supplementary material, which is available to authorized users.

A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum

“…This method is applicable to a wide range of fluorescent dyes; however, the readily available sulfo-cyanine5 was employed based on its ease of conjugation to the ligand and suitability of the analytical detection. The straightforward utilization of this NanoTemper technology was previously shown in the analysis of antibody binding affinities to drugs 17 and in quantitative analysis of different biomolecular interactions 37 by the microscale thermophoresis (MST) assay. This proposed method was compared with the previously reported ED-UPLC-MS/MS.…”

“…The viability of using ED-fluorimetry in determining the antibody binding-site concentration was demonstrated using the in-house-generated 6-AmHap-mAb with hapten-fluorophore tracers, 6-AmHap-Cy5 and morHap-Cy5 ( Scheme 1 A,C). The binding affinities of 6-AmHap-mAb against 6-AmHap-Cy5 and morHap-Cy5 established by a conventional MST assay 17 were found to be 0.65 ± 0.28 and 0.15 ± 0.03 pM, respectively. There is no significant difference ( p = not significant; paired T -test) in the binding affinities of 6-AmHap-mAb to both hapten-fluorophore tracers.…”

Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice

“…The heroin hapten, 6-AmHap, and the fentanyl hapten, para -AmFenHap, were separately coupled to the tetanus toxoid (TT) carrier protein using the optimized coupling method as previously described. ,,, Briefly, TT was incubated with the NHS-(PEG) 2 -maleimide cross-linker [SM­(PEG) 2 ] cross-linker at a 1:1600 molar ratio for 2 h. Excess linker was removed using a Zeba spin column, and the protein content of the eluate was quantified using a bicinchoninic acid (BCA) assay kit. The excess haptens were removed by repeated dialysis against phosphate buffered saline (PBS), pH 7.4, at 4 °C.…”

Army Liposome Formulation (ALF) family of vaccine adjuvants

“…A candidate synthetic hapten (6-AmHap) was conjugated to tetanus toxoid as a carrier protein, and when tested either with ALF or ALFA, high titers of antibodies to heroin and heroin degradation products were achieved. The antibodies also cross-reacted with several other relevant opioids, such as oxycodone and hydrocodone [82]. When challenged intravenously with heroin, the rodent antibodies blocked the characteristic physiological effects of heroin [81].…”