Detection and monitoring PLA 2 R autoantibodies by LIPS in membranous nephropathy.

Autoantibodies against the M-type phospholipase A2 receptor (PLA2 R) are specific markers for primary membranous nephropathy (MN). Quantification of PLA2 R 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 PLA2 R autoantibodies. For these studies, a cDNA fragment encoding the first 858 amino acids of PLA2 R 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 PLA2 R-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 PLA2 R autoantibody levels in a subset of patients with primary MN and demonstrated 100% sensitivity compared to ELISA and/or Western blotting. The PLA2 R-NanoLuc LIPS assay demonstrated 100% specificity matching the ELISA, but the specificity of the PLA2 R-Gluc LIPS assays was slightly lower (97%). Further analysis revealed that autoantibody levels determined by PLA2 R-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 PLA2 R-NanoLuc assay for diagnosis and management of MN.