title: An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.
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.
authors: Division of Nephrology, Hannover Medical School, Hannover, Germany.
authors: Astrid
authors: A
authors: Behnert
authors: Marvin J
authors: MJ
authors: Fritzler
authors: Beina
authors: B
authors: Teng
authors: Meifeng
authors: M
authors: Zhang
authors: Frank
authors: F
authors: Bollig
authors: Hermann
authors: H
authors: Haller
authors: Andrej
authors: A
authors: Skoberne
authors: Michael
authors: M
authors: Mahler
authors: Mario
authors: M
authors: Schiffer
mesh_terms: D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000032:analysis / Q000276:immunology; D001324:Autoantigens / Q000737:chemistry* / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D015433:Glomerulonephritis, Membranous / Q000175:diagnosis*; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007834:Lasers; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017434:Protein Structure, Tertiary; D054507:Receptors, Phospholipase A2 / Q000737:chemistry* / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D010446:Peptide Fragments; D054507:Receptors, Phospholipase A2
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