Table2_Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry.XLSX

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（monoclonal antibodies, mAbs）作为免疫测定中的核心分析组分，被广泛应用于临床诊断、食品分析与药物发现等领域的靶标分子检测。通常将官能团偶联至赖氨酸或半胱氨酸残基，以辅助单克隆抗体的固定，或是实现其在抗体-抗原复合物中的检测。免疫测定的优异性能，依赖于单克隆抗体对抗原的亲和力与特异性。然而，偶联反应可能引发高阶结构（higher order structural, HOS）变化，最终影响免疫测定的整体性能。本研究选取4种不同偶联方式的单克隆抗体作为模型体系，并通过质谱法对其开展全面表征。具体而言，采用液相色谱-质谱联用法（liquid-chromatography mass-spectrometry, LC-MS）对完整蛋白进行分析，以明确偶联的取代程度与分布情况；开展氢氘交换质谱法（hydrogen deuterium exchange mass-spectrometry, HDX-MS）实验，对偶联后单克隆抗体的空间结构进行表征；同时实施免疫测定实验，以监测偶联反应对所选抗体结合特性的影响。研究发现，质谱分析结果与结合实验结果具有高度一致性。尤其值得关注的是，抗体的整体结构柔性在半胱氨酸偶联后显著提升，而在赖氨酸偶联后则明显下降。带有体积庞大官能团的单克隆抗体偶联物，因诱导产生的空间位阻效应，往往会降低氘摄入动力学速率。总体而言，本研究揭示了免疫测定抗体的偶联过程、分子结构与功能活性之间的内在关联，并证实了质谱技术有助于加深对偶联反应的理解，同时提供可用于预测免疫测定性能的关键见解。