Preparation of an epitope-based recombinant diagnostic antigen specific to anti-phospholipase A(2) receptor 1 antibodies

BACKGROUND: According to recent studies, the phospholipase A2 receptor 1 (PLA(2)R1) may be used as a biomarker to diagnose idiopathic membranous nephropathy (iMN). Moreover, the immune-dominant regions of PLA(2)R1 have been identified. The aim of the present study was to construct a diagnostic antigen based on the immune-dominant region of PLA(2)R1 and develop a specific serological detection method for PLA(2)R1 antibodies. RESULTS: The tandem multi-epitope diagnostic antigen (designated ‘R101’), which includes aa 39–130 (CysR), aa 238–356 (CTLD1), and aa 1136–1234 (CTLD7) of PLA(2)R1; thioredoxin at the N-terminus; and a His tag at the C-terminus, was prepared at a concentration of 2.36 mg/mL and purity of 97.32% using Escherichia coli expression and affinity and anion exchange chromatography purification. The integrity and antigenicity of the R101 protein was demonstrated by western blot analysis using anti-Trx, anti-His, and anti-PLA(2)R1 monoclonal antibodies as the primary antibodies. By analysing 120 positive serum samples identified by biopsy-proven iMN (gold standard) and 240 negative samples identified by an established ELISA based on R101 protein, we concluded that the cut-off value, kappa value, sensitivity, specificity, and agreement rate were 0.305, 0.881, 91.67, 96.25, and 94.72% respectively. The receiver operating characteristic (ROC) curve illustrated that the diagnostic accuracy and practicability of the ELISA was excellent. The area under the curve was 0.986. CONCLUSIONS: Using prokaryotic expression and chromatography purification, immune-dominant regions of PLA(2)R1 with excellent antigenicity can be prepared and applied to serological detection of PLA(2)R1 antibodies.