Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy

Background: Autoantibodies against the M-type phospholipase A2 receptor (PLA(2)R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA(2)R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using recombinant PLA(2)R1 as a substrate has been widely available but lacks a finely graduated assessment of antibody concentrations. Methods: In order to setup a standardized ELISA, the extracellular domain of human PIA(2)R1 was expressed in HEK293. The purified protein was used to form the solid-phase in an ELISA which was then employed to analyze sera from 200 patients with primary MN, 27 patients with secondary MN, 230 patients with other glomerular diseases, 316 patients with systemic autoimmune diseases, and from 291 healthy blood donors. Results: At a set specificity of 99.9% the sensitivity of the anti-PLA(2)R1 IgG ELISA was found to be 96.5%. A similar sensitivity (98.5%) was obtained when binding of only subclass IgG(4) was analyzed. The calibrated assay showed a good class correlation with the results of the RC-IFA, was robust and could be stored for several months without any loss of quality. Conclusion: The results demonstrate that the new test system is qualified for routine use and that it has an almost perfect agreement with both, the clinical characterization of the patients and the results generated with RC-IFA. (C) 2013 Elsevier B.V. All rights reserved.