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 abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

中文翻译：

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一种基于溶液的快速方法，用于确定海洛因半抗原诱导的抗体与海洛因，其代谢物和其他阿片类药物的亲和力。

我们首次描述了一种利用微尺度热泳（MST）技术确定多克隆抗体与小分子亲和力的方法。使用新型异源MST，我们已经精确地测量了血清抗体对海洛因的基于溶液的结合亲和力，这在以前是其他当前可用的方法所无法实现的。此外，这种失配方法（即使用交叉反应的半抗原示踪剂）在文献中从未报道过。与平衡透析结合超高效液相色谱/串联质谱（ED-UPLC / MS / MS）进行比较时，这种新颖的MST方法对主要海洛因代谢物6-AM和吗啡的多克隆抗体产生相似的结合亲和力值。此外，我们在此报告了这种新型交叉反应半抗原的合成方法，MorHap-乙酰胺-一种用于研究海洛因半抗原与抗体相互作用的有用类似物。使用异源MST，我们能够确定对各种滥用阿片类药物的多克隆抗体的亲和力，直至纳摩尔精度。在优化此方法的同时，我们进一步发现，这些抗体的存在以浓度依赖的方式保护海洛因免受血清酯酶降解。最后，使用许多结构上不同的阿片类药物的亲和力数据，我们能够剖析对抗体结合至关重要的部分。本文介绍的新型MST方法可以扩展到易于降解的任何配体的分析，不仅可以用于开发针对滥用物质的疫苗，还可以应用于分析药物中的小分子/蛋白质相互作用。血清的存在。图形化使用微尺度热泳确定半抗原诱导的抗体亲和力的策略。