Development of a Time-Resolved Fluorescence Immunoassay for Quantitative Detection of Obinutuzumab and Its Application in Monitoring Membranous Nephropathy Treatment

Given the growing clinical application of obinutuzumab in membranous nephropathy (MN) and the absence of reliable quantitative assays for therapeutic drug monitoring, this study aimed to develop and validate a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of obinutuzumab in human serum. The TRFIA utilized a biotin–streptavidin system with an idiotype-specific capture antibody and europium-labeled anti-human IgG for signal detection. Assay conditions were optimized, and analytical performance was evaluated in terms of linearity, sensitivity, precision, recovery, specificity, and comparability with an enzyme-linked immunosorbent assay (ELISA). The assay showed a linear range of 0.5–200 ng/mL, with a limit of detection of 0.023 ng/mL and a limit of quantification of 0.16 ng/mL. Intra- and inter-assay coefficients of variation were below 9%, and recovery ranged from 93.9% to 104.9%. No cross-reactivity was observed with other therapeutic monoclonal antibodies. Compared with ELISA, the TRFIA demonstrated approximately tenfold higher sensitivity and a broader linear dynamic range, with a strong correlation between the two methods (R2 = 0.98). In patients receiving obinutuzumab, serum drug concentrations exhibited substantial inter-individual variability, and longitudinal monitoring revealed a time-dependent decline consistent with expected pharmacokinetic behavior. This TRFIA method enables accurate and sensitive monitoring of serum obinutuzumab levels in MN patients and may provide a useful tool for therapeutic drug monitoring and individualized treatment strategies.