Dual-antigen sandwich-type time-resolved fluoroimmunoassay for phospholipase A2 receptor antibody, a new marker of membranous nephropathy

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) method for detecting M-type phospholipase A2 receptor (PLA2R) antibody and to investigate the diagnostic value of serum PLA2R antibody for idiopathic membranous nephropathy (IMN). Methods The microplates coated with recombined PLA2R antigen and Eu3+ -streptavidin labeled PLA2R antigen were used to establish a dual-antigen sandwich-type TRFIA for PLA2R antibody detection (anti-PLA2R-TRFIA). The serum concentrations of PLA2R antibody in 63 IMN patients (36 males, 27 females, age 25-75 years) and 90 healthy volunteers (30 males, 60 females, age 22-53 years) were quantitatively analyzed. Kruskal-Wallis H test and Mann-Whitney u test were used to analyze the data. Results The range of anti-PLA2R-TRFIA was 0-10 mg/L and the sensitivity was 5 μg/L, while ED20, ED50 and ED80 of the standard curve were (0.144±0.012), (0.707±0.029) and (3.466±0.098) mg/L, respectively. The CV of inter- and intra-assay were 4.7% and 5.1%, respectively. The average concentration of serum PLA2R antibody in healthy volunteers was 0.455(0.320, 0.593) mg/L, but in IMN patients it was 2.690(0.717, 7.750) mg/L (z=-3.688, P<0.05). Meanwhile, the serum levels of PLA2R antibody in IMN patients were significantly different between different stages and ages (χ2 values: 10.328, 9.716, both P<0.05). According to the receiver operating characteristic (ROC) curve, when the diagnostic cut-off was set at 0.80 mg/L for IMN detection, the sensitivity and specificity of anti-PLA2R-TRFIA were 73.0%(46/63) and 95.6%(86/90), respectively. Conclusions Anti-PLA2R-TRFIA has been well established. This quick and easy-performance method could increase the diagnostic accuracy for IMN. Key words: Glomerulonephritis, membranous; Receptors, phospholipase A2; Fluoroimmunoassay