Development of a time-resolved fluoroimmunoassay for rituximab and its application to therapeutic drug monitoring

Background: Rituximab pharmacokinetics in patients with membranous nephropathy (MN) exhibit significant interindividual variability. Accurate measurement of serum rituximab concentrations is essential for effective therapeutic monitoring. This study develops a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for rituximab (rituximab-TRFIA) with a wide detection range, aimed at enhancing therapeutic drug monitoring in MN treatment.

Methods: A capture -type rituximab-TRFIA was developed using streptavidin-coated microplates, biotinylated anti-rituximab idiotypic antibodies, and Eu³⁺-labeled mouse anti-human IgG targeting the Fc fragment of rituximab. The assay was used to measure rituximab serum concentrations in MN patients treated with rituximab.

Results: The linear range of rituximab-TRFIA is 0.50 to 2500 ng/mL, with a limit of detection (LOD) of 0.062 ng/mL. The intra-assay coefficient of variation (CV) ranges from 1.97 % to 9.50 %, while the inter-assay CV ranges from 7.44 % to 9.99 %. The recovery rate ranges from 99.14 % to 107.75 %. No cross-reactivity was observed with other monoclonal antibody drugs (mAbs). The detection range is two orders of magnitude higher, and the sensitivity is six times greater than that of the enzyme-linked immunosorbent assay (ELISA). Rituximab-TRFIA showed strong consistency within the same measurement range compared to ELISA (P < 0.0001). The serum rituximab levels in MN patients were significantly higher than those in the control group (P < 0.0001). Among patients who did not achieve remission, 60 % to 70 % had insufficient drug concentrations, and rituximab pharmacokinetics followed the expected trends.

Conclusions: The rituximab-TRFIA provides high sensitivity, a wide detection range, and reliable performance for monitoring serum rituximab concentrations in MN patients, supporting personalized treatment strategies.