An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor

PLoS One. 2013 Apr 29;8(4):e61669. doi: 10.1371/journal.pone.0061669. Print 2013.

Abstract

The phospholipase A2 receptor (PLA2R) 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 PLA2R 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 PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R 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-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Autoantibodies / analysis
  • Autoantibodies / immunology
  • Autoantigens / chemistry*
  • Autoantigens / immunology*
  • Epitope Mapping / methods*
  • Glomerulonephritis, Membranous / diagnosis*
  • HEK293 Cells
  • Humans
  • Immunoassay / methods*
  • Lasers
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / immunology
  • Protein Structure, Tertiary
  • Receptors, Phospholipase A2 / chemistry*
  • Receptors, Phospholipase A2 / immunology*

Substances

  • Autoantibodies
  • Autoantigens
  • Peptide Fragments
  • Receptors, Phospholipase A2

Grant support

A.B. has been supported by the German Academic Exchange Service (DAAD), the Biomedical Sciences Exchange Program (BMEP) and StrucMed, Medical School Hanover. M.S. is supported by DFG grants (SCHI587/4–6). M.J.F. holds the Arthritis Research Chair at the University of Calgary and this work was supported by funds derived from that endowment. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.