Darwish Ibrahim A, Zhang Daohong, Alsalhi Mohammed S
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia.
College of Food Engineering, Bio-Nanotechnology Research Institute, Ludong University, Yantai, 264025, Shandong, China.
Heliyon. 2024 Jul 14;10(14):e34611. doi: 10.1016/j.heliyon.2024.e34611. eCollection 2024 Jul 30.
Zolbetuximab (ZOL) is a groundbreaking monoclonal antibody targeting CLDN 18.2, a cancer cell surface protein. It is a first-in-class therapy for gastric and gastroesophageal junction adenocarcinoma. However, there is currently any immunoassay available for bioanalysis of ZOL, hindering its pharmacokinetic studies, therapeutic monitoring, and safety profile refinement. To address this gap, this study presents the development and validation of a novel highly sensitive inner filter effect-based fluorescence immunoassay (IFE-FIA) with quantum dots (QDs) as a probe. This assay enables the quantitative determination of ZOL in plasma samples. The assay involved non-competitive capturing of ZOL from the samples using a specific antigen (CLDN 18.2 protein) immobilized on assay plate microwells. A horseradish peroxidase (HRP)-labelled anti-human IgG was used to measure the immune complex. The assay's detection system relies on the formation of a light-absorbing colored product through an HRP-catalyzed oxidative reaction with the substrate 3,3',5,5'-tetramethylbenzidine. This light absorption efficiently quenched the fluorescence of QDs via the IFE. The measured fluorescence signals corresponded to the concentrations of ZOL in the samples. The conditions of the IFE-FIA and its detection system were refined, and the optimum procedures were established. Following the guidelines of immunoassay validation for bioanalysis, the assay was validated, and all the validation criteria were acceptable. The assay demonstrates high sensitivity, accurately quantifying ZOL at concentrations as low as 10 ng/mL in plasma samples, with acceptable precision. Importantly, it avoids interferences from endogenous substances and plasma matrix. The recoveries in spiked human plasma ranged from 96.8 % to 104.5 %, with relative standard deviations of 4.1 %-6.5 %. The proposed IFE-FIA represents a valuable tool for quantifying ZOL in clinical settings, enabling assessment of its pharmacokinetics, therapeutic drug monitoring, and safety profile refinement.
佐贝妥昔单抗（ZOL）是一种开创性的单克隆抗体，靶向癌细胞表面蛋白CLDN 18.2。它是用于治疗胃及胃食管交界腺癌的同类首创疗法。然而，目前尚无用于佐贝妥昔单抗生物分析的免疫测定方法，这阻碍了其药代动力学研究、治疗监测以及安全性概况的优化。为填补这一空白，本研究介绍了一种新型的基于内滤光片效应的高灵敏度荧光免疫测定法（IFE - FIA）的开发与验证，该方法以量子点（QD）作为探针。此测定法能够定量测定血浆样本中的佐贝妥昔单抗。该测定法包括使用固定在测定板微孔上的特异性抗原（CLDN 18.2蛋白）从样本中竞争性捕获佐贝妥昔单抗。使用辣根过氧化物酶（HRP）标记的抗人IgG来检测免疫复合物。该测定法的检测系统依赖于通过HRP催化与底物3,3',5,5'-四甲基联苯胺发生氧化反应形成吸光有色产物。这种光吸收通过内滤光片效应有效地淬灭了量子点的荧光。测得的荧光信号与样本中佐贝妥昔单抗的浓度相对应。对IFE - FIA及其检测系统的条件进行了优化，并确定了最佳程序。按照生物分析免疫测定验证指南，对该测定法进行了验证，所有验证标准均符合要求。该测定法具有高灵敏度，能够在血浆样本中低至10 ng/mL的浓度下准确地定量佐贝妥昔单抗，精密度良好。重要的是，它避免了内源性物质和血浆基质的干扰。在加标的人血浆中的回收率为96.8%至104.5%，相对标准偏差为4.1% - 6.5%。所提出的IFE - FIA是在临床环境中定量佐贝妥昔单抗的一种有价值的工具，有助于评估其药代动力学、治疗药物监测以及安全性概况的优化。
