Detection and monitoring PLA2R autoantibodies by LIPS in membranous nephropathy.
Autoantibodies against the M-type phospholipase A2receptor (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.
Dental Clinical Research Core, NIDCR, NIH, Bethesda, MD, United States. Electronic address: burbelop@nidcr.nih.gov.
Peter D
PD
Burbelo
Department of Medicine, Section of Nephrology, Boston University School of Medicine, Boston, Mass, United States.
Laurence H
LH
Beck
Kidney Disease Branch, NIDDK, NIH, Bethesda, MD, United States.
Meryl
M
Waldman
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D019556:COS Cells; D016022:Case-Control Studies; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter; D015433:Glomerulonephritis, Membranous / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000628:therapy; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D008156:Luciferases / Q000235:genetics; D011237:Predictive Value of Tests; D054507:Receptors, Phospholipase A2 / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012008:Recurrence; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection; D016896:Treatment Outcome
D001323:Autoantibodies; D015415:Biomarkers; D054507:Receptors, Phospholipase A2; D011993:Recombinant Fusion Proteins; D008156:Luciferases