Fc-tagged PLA2R antigen enables high-performance chemiluminescent detection for non-invasive idiopathic membranous nephropathy diagnosis

• Full-length PLA2R Fc fusion protein achieved 100 % sensitivity and specificity. • pH 6.0 MES buffer coating enhancing signal-to-noise ratio by 45.08. • Polyclonal goat anti-human IgG improves sensitivity for low-titer samples. • Superior diagnostic accuracy and enhanced sensitivity/specificity v.s. ELISA kit. • High stability at RT and 4 °C, supporting widespread clinical adoption. Idiopathic membranous nephropathy (IMN) is characterized by anti-phospholipase A2 receptor (PLA2R) antibodies, yet current detection methods lack sensitivity or are invasive. We developed a chemiluminescent assay to address these limitations. Three PLA2R antigens (Fc-tagged full, His-tagged, D3 core segment) were expressed in Expi293E cells and purified. Goat/mouse anti-human IgG antibodies were compared for detection efficiency. Kit stability was evaluated at 4 °C and 37 °C. Clinical validation included 75 biopsy-confirmed cases (50 IMN, 25 non-IMN), comparing the kit’s performance against a commercial ELISA. The PLA2R-Fc antigen demonstrated 100 % sensitivity and specificity, outperforming His-tagged (100 % sensitivity, 97 % specificity) and D3-truncated variants (60 % sensitivity). Coating in pH 6.0 MES buffer achieved the highest signal-to-noise ratio (45.08 vs. 25.76 in HEPES). Goat anti-human IgG enabled lower detection limits (22.5 U mL −1 vs. 45 U mL −1 for monoclonal antibody). The kit retained stable over 12 months at 4 °C. In clinical testing, the chemiluminescent assay showed superior concordance with histopathology, higher sensitivity, and specificity, particularly for low-titer samples. This chemiluminescent anti-PLA2R antibody assay combines high sensitivity, specificity, and stability, enabling non-invasive IMN diagnosis and therapeutic monitoring. Its technical optimizations address key limitations of existing methods, offering a reliable diagnostic tool for IMN. In this study, the high sensitivity, specificity and stability of PLA2R chemiluminescence kit was successfully developed