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Detection and monitoring PLA2R autoantibodies by LIPS in membranous nephropathy
Peter D Burbelo1, Laurence H Beck2, Meryl Waldman3
1Dental Clinical Research Core, NIDCR, NIH, Bethesda, MD, United States.
Abstract:
Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are specific markers for primary membranous nephropathy (MN). Quantification of PLA2R 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 PLA2R autoantibodies. For these studies, a cDNA fragment encoding the first 858 amino acids of PLA2R 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 PLA2R-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 PLA2R autoantibody levels in a subset of patients with primary MN and demonstrated 100% sensitivity compared to ELISA and/or Western blotting. The PLA2R-NanoLuc LIPS assay demonstrated 100% specificity matching the ELISA, but the specificity of the PLA2R-Gluc LIPS assays was slightly lower (97%). Further analysis revealed that autoantibody levels determined by PLA2R-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 PLA2R-NanoLuc assay for diagnosis and management of MN.
Insights
A new assay detects autoantibodies against the phospholipase A2 receptor (PLA2R), crucial for diagnosing and monitoring primary membranous nephropathy (MN). The PLA2R-NanoLuc LIPS assay shows high accuracy and potential for clinical management.
Area of Science:
- Nephrology
- Immunology
- Biotechnology
Background:
- Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are specific biomarkers for primary membranous nephropathy (MN).
- Quantifying PLA2R autoantibodies is essential for noninvasive diagnosis and monitoring of MN.
- Existing diagnostic methods may have limitations in sensitivity or specificity.
Purpose of the Study:
- To develop and validate a highly quantitative luciferase immunoprecipitation systems (LIPS) assay for detecting PLA2R autoantibodies.
- To assess the diagnostic accuracy and clinical utility of the LIPS assay compared to established methods.
Main Methods:
- Cloning of a cDNA fragment encoding the first 858 amino acids of PLA2R protein.
- Generation of N-terminal antigen fusion constructs with Gaussia luciferase (Gluc) and Nano luciferase (NanoLuc) reporters.
- Testing of recombinant PLA2R-luciferase fusion proteins using LIPS in healthy controls, patients with other kidney diseases, and patients with MN.
Main Results:
- LIPS assays with both Gluc and NanoLuc reporters detected robust PLA2R autoantibody levels in primary MN patients.
- The PLA2R-NanoLuc LIPS assay demonstrated 100% sensitivity and 100% specificity, matching ELISA.
- PLA2R-NanoLuc LIPS assay results correlated strongly with urinary protein excretion and disease activity, and were sensitive for detecting relapse.
Conclusions:
- The developed LIPS assay, particularly the PLA2R-NanoLuc variant, is a highly quantitative and accurate method for detecting PLA2R autoantibodies.
- This assay holds significant potential for the diagnosis and clinical management of primary membranous nephropathy.
- The assay's ability to correlate with disease activity and detect relapse underscores its utility in patient monitoring.
