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

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.

Lethal Hemorrhagic Disease and Clinical Illness Associated with Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins

“…Preparation of Ruc antigen fusion constructs. Codon-optimized sequences were synthesized (GenScript) for predicted EEHV proteins and cloned in frame with the Gluc gene in the mammalian expression vector pGAUS3 (25,26). EEHV1A U39, U14, U28, and ORF-Q (clade A) proteins were derived from EEHV1A Kimba (27) and the EEHV4 and EEHV5 U39 proteins from Vijay (4) and Baylor (28) genome sequences, respectively.…”

Rapid, Quantitative, and High-Sensitivity Detection of Anti-Phospholipase A2 Receptor Antibodies Using Novel Cdse/Zns-Based Fluorescence Immunosorbent Assay

“…Previous studies used western blotting, which requires electrophoresis and immunoblotting, as well as the use of naturally puri ed glomerular glycoprotein or extracts from cells overexpressing PLA2R, which may not be available in conventional clinical laboratories. [26,29], ALBIA (51.5-66.9%) [18,30], TRFIA (71.0%) [28], LIPS (53.3%) [20], and ChLIA (83.9%) [21]]. Although the positive rates of western blotting (53.0-81.7%) [31,32] and CBA-IFA (48.0-82.3%) [33,34] are also comparable to those of quantitative assays, the semiquantitative nature of these methods hinders their clinical application.…”

“…However, in ELISA, signal generation is based on time-dependent enzyme reactions and an enzyme is susceptible to environmental factors and highly vulnerable to the in uence of matrix components [17]. Recently, an addressable laser bead immunoassay (ALBIA) [18], timeresolved uoroimmunoassays (TRFIAs) [19], luciferase immunoprecipitation systems (LIPS) [20], and a chemiluminescence immunoassay (ChLIA) [21] were reported for the quantitative detection of anti-PLA2R antibodies. Although these methods have contributed signi cantly toward the detection PLA2R antibodies, the detection equipment are not su ciently portable, and the detection time is not su ciently rapid.…”

Autoantibodies are present before the clinical diagnosis of systemic sclerosis

“…Luciferase immunoprecipitations systems (LIPS) is a fluid-phase immunoassay that utilizes luciferase-tagged recombinant antigens to detect antibodies against linear and conformational epitopes of infectious and autoimmune target proteins. We and others have found LIPS to demonstrate high diagnostic performance for detecting autoantibodies in a number of different autoimmune conditions [ 22 ] including Sjögren’s syndrome [ 23 ], Type I diabetes [ 24 ], systemic lupus erythematosus [ 25 ], autoimmune gastritis [ 26 ], membranous nephropathy [ 27 ], and APECED [ 28 , 29 ]. In several of these studies, LIPS elucidated unique patient autoantibody profiles [ 23 – 25 , 28 ] that potentially associated with disease subsets and/or autoimmune symptoms.…”

“…In several of these studies, LIPS elucidated unique patient autoantibody profiles [ 23 – 25 , 28 ] that potentially associated with disease subsets and/or autoimmune symptoms. LIPS with its wide dynamic range of detection and low background has been highly useful for monitoring changes in antibody levels in longitudinal serum samples in both infectious [ 30 ] and autoimmune diseases [ 27 ]. Here we report our exploratory study profiling autoantibodies from the serum of SSc subjects obtained before clinical disease diagnosis and assess whether unique autoantibody responses might be associated with future onset of SSc/SRC.…”