The Atomic Medicine Initiative, University of Technology Sydney, 15 Broadway, Sydney, NSW, 2007, Australia.
Anal Bioanal Chem. 2019 Jun;411(16):3553-3560. doi: 10.1007/s00216-019-01836-9. Epub 2019 Apr 27.
This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies' integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7 min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing Eu-labelled polyclonal antibodies. Graphical abstract.
本研究提出了一种新的尺寸排阻色谱-电感耦合等离子体质谱（SEC-ICP-MS）方法，用于对镧系标记抗体的免疫分析物进行特征描述和定量。SEC-ICP-MS 与双同位素稀释法相结合，可轻松验证抗体的完整性、确定批间标记效率、监测每个标记步骤，并在与靶蛋白孵育后定量免疫复合物。在动态反应池中添加氧可通过质量转移（LOD = 3.7 ng/mL）改善对作为抗体和靶蛋白标记物的硫的检测，同时保持对镧系元素分析的足够灵敏度。超高效液相色谱（UHPLC）SEC 确保了一种快速色谱方法，标记和未标记的抗体、免疫复合物以及用于镧系标记抗体的未共轭聚合物的分离时间均在 7 分钟以下。SEC 校准估计了所有峰的分子量，并为免疫化学反应以及反应物和产物的化学计量学提供了有价值的见解。一种新的在线同位素稀释分析（IDA）能够实现硫和镧系信号以及感兴趣的蛋白质的绝对定量。免疫复合物和标记抗体的色谱分离允许从单个质量流色谱图同时测定抗体/金属化学计量比和靶蛋白浓度。在与 Eu 标记的初级多克隆抗体孵育后，对免疫球蛋白蛋白进行了定量。通过直接标记目标蛋白 Gd 进行平行、同时定量来验证该程序。通过直接标记蛋白质确定的浓度与采用 Eu 标记的多克隆抗体的免疫化学方法相差 1.9%。
