Development and preliminary clinical application of a time-resolved fluoroimmunoassay for anti-rituximab antibodies in membranous nephropathy

Objective In some patients with membranous nephropathy (MN) receiving rituximab therapy, treatment failure may be partly related to the development of anti-rituximab antibodies (ADAs). This study aimed to establish a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for detecting ADAs in these patients.Method A streptavidin-coated microplate was used as the solid phase, with biotinylated rituximab as the capture antibody and Eu3+ -labeled rituximab as the detection antibody. A bridging assay was developed to detect ADAs, and the method was optimized, validated, and preliminarily applied in clinical testing.Results The developed TRFIA exhibited a linear detection range of 12.5 ng/mL to 800 ng/mL, with a detection limit (LOD) of 3.93 ng/mL. The intra‑assay coefficient of variation (CV) ranged from 3.07% to 7.90%, and the inter‑assay CV ranged from 2.57% to 11.32%. Recovery rates spanned 87.06% to 104.11%, and no cross‑reactivity was detected with anti‑obinutuzumab or anti‑eculizumab. Compared to enzyme‑linked immunosorbent assay (ELISA), the TRFIA showed superior sensitivity (3.9 ng/mL vs. 10 ng/mL) and maintained strong agreement in sample detection (p < 0.0001).Conclusion This assay demonstrates specific potential clinical utility in monitoring resistance to rituximab, providing valuable guidance for optimizing anti-CD20 monoclonal antibody therapy and developing personalized treatment plans.