A novel highly sensitive inner filter effect-based fluorescence immunoassay with quantum dots for bioanalysis of zolbetuximab, a monoclonal antibody used for immunotherapy of gastric and gastroesophageal junction adenocarcinoma.

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.