Detection and monitoring PLAR autoantibodies by LIPS in membranous nephropathy.

Autoantibodies against the M-type phospholipase A receptor (PLAR) are specific markers for primary membranous nephropathy (MN). Quantification of PLAR autoantibodies is an important, noninvasive tool that facilitates the diagnosis and monitoring of primary MN. In this report we describe a highly quantitative luciferase immunoprecipitation systems (LIPS) assay for detecting PLAR autoantibodies. For these studies, a cDNA fragment encoding the first 858 amino acids of PLAR protein was cloned to generate N-terminal antigen fusion constructs with Gaussia luciferase (Gluc) and Nano luciferase (NanoLuc) reporters. Following transfection, crude cell extracts containing the recombinant PLAR-luciferase fusion proteins were tested by LIPS on healthy controls, subjects with other kidney disease and subjects with MN. LIPS testing with both reporters detected robust PLAR autoantibody levels in a subset of patients with primary MN and demonstrated 100% sensitivity compared to ELISA and/or Western blotting. The PLAR-NanoLuc LIPS assay demonstrated 100% specificity matching the ELISA, but the specificity of the PLAR-Gluc LIPS assays was slightly lower (97%). Further analysis revealed that autoantibody levels determined by PLAR-NanoLuc LIPS correlated well with urinary protein excretion (R=0.79) and disease activity and was very sensitive for detecting clinical relapse. These results highlight the potential utility of the LIPS PLAR-NanoLuc assay for diagnosis and management of MN.