Development and application of an ultrasensitive immunoassay for antiphospholipase A2 receptor antibodies in the differential diagnosis of membranous nephropathy†

Objective: to develop an ultrasensitive time-resolved fluoroimmunoassay (TRFIA) for anti-PLA2R IgG to improve serological diagnosis of primary membranous nephropathy (PMN). The three-step indirect TRFIA uses signal cascade amplification to enhance sensitivity while maintaining specificity, aiming to increase positive detection rates in PMN patients and evaluate analytical performance and clinical utility in 65 PMN patients (including 34 patients with negative Elisa test values), disease controls, and healthy controls. Results: the ultrasensitive PLA2R-IgG-TRFIA had a sensitivity of 0.44 RU per mL, a wide linear range (2.3–550 RU per mL), low intra- (3.16–5.76%) and inter-assay (4.39–9.19%) CVs, and acceptable recoveries (95.48–107.54%). A cutoff of 4.28 RU per mL ensured 100% specificity. It detected anti-PLA2R IgG in 26 of the 34 ELISA-negative PMN sera, consistent with pathology. Conclusion: the ultrasensitive PLA2R-IgG-TRFIA method for detecting anti-PLA2R IgG achieves higher sensitivity and specificity compared to ELISA, thus increasing the positive detection rate of PMN.