Department of Pharmacology, Physiology, and Neurobiology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH, 45267-0575, USA.
Department of Pharmacology, Physiology, and Neurobiology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH, 45267-0575, USA.
Anal Biochem. 2025 Jan;696:115690. doi: 10.1016/j.ab.2024.115690. Epub 2024 Oct 18.
Biotinylation of proteins, including antibodies, is a very useful and important modification for a variety of biochemical characterizations, including anti-drug antibody (ADA) assays used to detect antibodies raised against therapeutic antibodies. We assessed different degrees of biotin labeling of an anti-cocaine mAb currently under development for treating cocaine use disorder. We noted that higher levels of biotin labeling dramatically decreased mAb solubility, and increased the tendency to bind to surfaces, complicating characterization of the biotinylated antibody. Specifically, in phosphate buffered saline, labeling stoichiometries of more than about 3 biotin/mAb resulted in decreased recoveries due to increased binding to surfaces and decreased mAb solubility. Native gel agarose electrophoresis, differential scanning fluorimetry, and isothermal titration calorimetry all demonstrated changes in the mAb which became more pronounced above a labeling ratio of 3 biotin/mAb. At 3.0 biotin/mAb, there were minimal changes in solubility, adsorptivity, exposure of hydrophobic dye-binding sites, heat stability, and cocaine binding, in stark contrast to labeling with 5.6 biotin/mAb. Thus, the degree of biotinylation should be kept at about 3 biotin/mAb to maintain antigen binding and general structure, solubility, and stability of this mAb, a finding which may be important for other similar mAbs currently in use or under development.
蛋白质的生物素化，包括抗体，是用于各种生化特性分析的非常有用和重要的修饰方法，包括用于检测针对治疗性抗体产生的抗体的抗药物抗体（ADA）测定。我们评估了目前正在开发用于治疗可卡因使用障碍的抗可卡因 mAb 的不同程度的生物素标记。我们注意到，更高水平的生物素标记大大降低了 mAb 的溶解度，并增加了与表面结合的趋势，从而使生物素化抗体的特性复杂化。具体来说，在磷酸盐缓冲盐水（PBS）中，由于表面结合增加和 mAb 溶解度降低，标记比约为 3 个生物素/mAb 导致回收率降低。天然凝胶琼脂糖电泳、差示扫描荧光法和等温滴定量热法都证明了 mAb 的变化，当标记比超过 3 个生物素/mAb 时，变化变得更加明显。在 3.0 个生物素/mAb 时，溶解度、吸附性、疏水性染料结合位点的暴露、热稳定性和可卡因结合的变化最小，与 5.6 个生物素/mAb 的标记形成鲜明对比。因此，生物素化的程度应保持在大约 3 个生物素/mAb，以维持抗原结合以及该 mAb 的一般结构、溶解度和稳定性，这一发现可能对目前正在使用或正在开发的其他类似 mAb 很重要。
