SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays
SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays
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DOI：
10.1007/s00216-019-01836-9
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发表时间：
2019-06-01
影响因子：
4.3
通讯作者：
Doble, Philip
中科院分区：
文献类型：
--
作者：
Clases, David;de Vega, Raquel Gonzalez;Doble, Philip
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法（SEC-ICP-MS）的表征和定量的免疫分析与镧系元素标记的抗体。SEC-ICP-MS结合双同位素稀释方法使得能够容易地验证抗体的完整性、确定批次间标记效率、监测每个标记步骤以及在与靶蛋白孵育后定量免疫复合物。向动态反应池中添加氧气改善了通过质量偏移（LOD=3.7ng/mL）作为抗体和靶蛋白的标记物的硫的检测，同时保持了镧系元素分析的足够灵敏度。超高效液相色谱（UHPLC）SEC确保了标记和未标记抗体、免疫复合物和用于镧系元素标记抗体的未缀合聚合物的分离时间在7分钟以下的快速色谱方法。SEC校准估计了所有峰的分子量，并在免疫化学反应和反应物和产物的化学计量中提供了有价值的见解。一种新的在线同位素稀释分析（IDA），使硫和镧系元素的信号和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/金属化学计量和靶蛋白浓度。在与Eu-153标记的一级多克隆抗体孵育后定量免疫球蛋白。该程序进行了验证与Gd-156直接标记的目标蛋白的平行，同时定量。通过直接标记的蛋白质确定的浓度偏离1.9%的免疫化学方法采用Eu-153标记的多克隆抗体。
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=3.7ng/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 7min 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-153-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with Gd-156 for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing Eu-153-labelled polyclonal antibodies.