Preparation of an epitope-based recombinant diagnostic antigen specific to anti-phospholipase A2 receptor 1 antibodies

Abstract

Abstract Background According to recent studies, the phospholipase A2 receptor 1 (PLA2R1) may be used as a biomarker to diagnose idiopathic membranous nephropathy (iMN). Moreover, the immune-dominant regions of PLA2R1 have been identified. The aim of the present study was to construct a diagnostic antigen based on the immune-dominant region of PLA2R1 and develop a specific serological detection method for PLA2R1 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 PLA2R1; 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-PLA2R1 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 PLA2R1 with excellent antigenicity can be prepared and applied to serological detection of PLA2R1 antibodies.

Keywords

• Phospholipase A2 receptor 1 (PLA2R1)
• Prokaryotic expression
• Chromatographic purification
• ELISA