Proteome Center Rostock, University Medicine Rostock and University of Rostock, Schillingallee 69, 18059 Rostock, Germany.
Waters GmbH|TA Instruments, Helfmann-Park 10, 65760 Eschborn, Germany.
Biomolecules. 2023 Jan 16;13(1):187. doi: 10.3390/biom13010187.
Antibody-based point-of-care diagnostics have become indispensable for modern medicine. In-depth analysis of antibody recognition mechanisms is the key to tailoring the accuracy and precision of test results, which themselves are crucial for targeted and personalized therapy. A rapid and robust method is desired by which binding strengths between antigens and antibodies of concern can be fine-mapped with amino acid residue resolution to examine the assumedly serious effects of single amino acid polymorphisms on insufficiencies of antibody-based detection capabilities of, e.g., life-threatening conditions such as myocardial infarction. The experimental ITEM-FOUR approach makes use of modern mass spectrometry instrumentation to investigate intact immune complexes in the gas phase. ITEM-FOUR together with molecular dynamics simulations, enables the determination of the influences of individually exchanged amino acid residues within a defined epitope on an immune complex's binding strength. Wild-type and mutated epitope peptides were ranked according to their experimentally determined dissociation enthalpies relative to each other, thereby revealing which single amino acid polymorphism caused weakened, impaired, and even abolished antibody binding. Investigating a diagnostically relevant human cardiac Troponin I epitope for which seven nonsynonymous single nucleotide polymorphisms are known to exist in the human population tackles a medically relevant but hitherto unsolved problem of current antibody-based point-of-care diagnostics.
基于抗体的即时诊断已成为现代医学不可或缺的手段。深入分析抗体识别机制是调整测试结果准确性和精度的关键，而测试结果的准确性和精度对于靶向和个性化治疗至关重要。人们希望有一种快速而稳健的方法，可以精细地确定抗原与抗体之间的结合强度，分辨率达到氨基酸残基水平，从而检查单个氨基酸多态性对抗体检测能力不足的假设严重影响，例如，危及生命的情况，如心肌梗死。实验中的 ITEM-FOUR 方法利用现代质谱仪器在气相中研究完整的免疫复合物。ITEM-FOUR 与分子动力学模拟相结合，可以确定在定义的抗原表位内个别交换的氨基酸残基对免疫复合物结合强度的影响。野生型和突变型表位肽根据它们相对于彼此的实验确定的离解焓进行排序，从而揭示出哪种单一氨基酸多态性导致抗体结合减弱、受损甚至完全丧失。研究一个具有诊断意义的人类心肌肌钙蛋白 I 表位，该表位在人类中已知存在七个非同义单核苷酸多态性，解决了当前即时抗体诊断中一个具有医学意义但尚未解决的问题。
