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.
Anal Bioanal Chem. 2018 Jun;410(16):3885-3903. doi: 10.1007/s00216-018-1060-4. Epub 2018 Apr 19.
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.
我们首次描述了一种利用微尺度热泳（MST）技术测定多克隆抗体与小分子亲和力的方法。使用一种新型的异源 MST，我们已经准确地测量了血清抗体对海洛因的基于溶液的结合亲和力，这是以前其他现有方法无法做到的。此外，这种错配方法（即使用交叉反应半抗原示踪剂）在文献中从未有过报道。与平衡透析结合超高效液相色谱/串联质谱法（ED-UPLC/MS/MS）相比，这种新型 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 MorHap-乙酰胺的合成方法，这对半抗原抗体相互作用研究非常有用。使用异源 MST，我们能够确定多克隆抗体对各种滥用阿片类药物的亲和力，精度可达纳摩尔级。在优化该方法的过程中，我们进一步发现，这些抗体以浓度依赖的方式保护海洛因免受血清酯酶的降解。最后，使用多种结构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。本文提出的新型 MST 方法可以扩展到分析任何容易降解的配体，不仅可以应用于滥用物质疫苗的开发，还可以应用于分析血清存在时小分子/蛋白质的相互作用。
