An Anti-Phospholipase A(2) Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

The phospholipase A(2) receptor (PLA(2)R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA(2)R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA(2)R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA(2)R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA(2)R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA(2)R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA(2)R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA(2)R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA(2)R.