Abstract
OBJECTIVE: This study aimed to develop a multiplex suspension assay for the simultaneous quantitative detection of anti-cysteine-rich domain (anti-CysR)/C-type lectin domain 1 (CTLD1)/C-type lectin domain 6-8 (CTLD678)-immunoglobulin G4 (IgG4) antibodies of M-type phospholipase A2 receptor (PLA2R) in the serum samples of patients to evaluate the clinical application value of PLA2R epitope spreading in disease prognosis. METHODS: CysR, CTLD1 and CTLD678 antigen domains were coupled to three types of microspheres. The optimal dilution ratio of biotinylated anti-human IgG4 was identified, and a multiplex suspension assay was evaluated in terms of linearity, sensitivity, precision, specificity and recovery rate. Lastly, the relationship between epitope spreading and the severity of idiopathic membranous nephropathy was evaluated. RESULTS: The content of all three epitopes could be detected simultaneously within 2 h. The intra-assay precision ranged from 4.74% to 9.48%, and the inter-assay precision was between 5.13% and 13.92%. Specific experiments showed that the human IgA antibody did not cause a cross-reaction. The recovery rates ranged between 90% and 100%. The cut-off values of epitopes CysR, CTLD1 and CTLD678 between healthy individuals and patients were 6.30, 12.38 and 10.06 Ru/mL, respectively. Among them, the positive rates of epitopes CysR, CTLD1 and CTLD678 were 100%, 34% and 75%, respectively. In addition, Group 3 (CTLD1 response) accounted for 73% of the 12 patients who were not in remission. Meanwhile, when the concentration of CTLD1 exceeds 42.76 Ru/mL, the patient's prognosis may be poorer. CONCLUSION: A multiplex suspension assay was developed for the simultaneous quantitative detection of anti-CysR/CTLD1/CTLD678-IgG4 antibodies. The epitope migration sequence of PLA2R molecules during disease progression may not follow a simple linear rule. Among them, the epitope CTLD1 is likely to exert the most significant influence on patient remission.