Luckau Luise, Groves Kate, Blencowe Chris, Scrimshaw Sam, Dent Alastair, Quaglia Milena
National Measurement Laboratory at LGC, Teddington, United Kingdom.
Fleet Bioprocessing Ltd., Hartley Wintney, United Kingdom.
Front Mol Biosci. 2022 Jul 7;9:866843. doi: 10.3389/fmolb.2022.866843. eCollection 2022.
Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance.
单克隆抗体（mAb）在免疫测定中被广泛用作分析成分，以检测临床诊断、食品分析和药物发现等应用中的目标分子。官能团通常与赖氨酸或半胱氨酸残基缀合，以帮助固定单克隆抗体或使其在抗体 - 抗原复合物中得以检测。良好的测定性能取决于单克隆抗体与抗原的亲和力和特异性。然而，缀合反应可能会导致高级结构（HOS）变化，并最终影响测定性能。在本研究中，选择了四种不同缀合的单克隆抗体作为模型系统，并通过质谱进行表征。特别地，通过液相色谱 - 质谱联用（LC - MS）进行完整蛋白质分析，以确定缀合的量和分布。进行氢氘交换质谱（HDX - MS）实验以对缀合的单克隆抗体进行结构表征。进行免疫测定实验以监测缀合对所选抗体结合特性的影响。发现质谱和结合实验结果之间具有良好的一致性。特别值得注意的是，半胱氨酸缀合后抗体的整体结构灵活性增加，而赖氨酸缀合后则降低。由于诱导的空间效应，单克隆抗体与庞大官能团的缀合往往会降低氘摄取动力学。总体而言，本研究表明了免疫测定抗体的缀合、结构和功能之间的相关性，以及质谱在增进对缀合反应的理解并提供可预测免疫测定性能的见解方面的益处。
