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.
Highlights
Membranous nephropathy (MN) is one of the most common causes of idiopathic nephrotic syndrome in adults [1,2]
The full-length human phospholipase A2 receptor (PLA2R) construct that was cloned into a green fluorescent protein (GFP) vector and over-expressed in HEK293 cells was verified by standard sequencing techniques
When addressable laser bead immunoassay (ALBIA) beads were indirectly coupled with the full length protein captured from cell lysates and tested with sera from 165 patients with membranous nephropathy; 85 (52%) of those tested positive, whereas 80 (48%) tested negative for anti-PLA2R antibodies on cell based assay (CBA)
Summary
Membranous nephropathy (MN) is one of the most common causes of idiopathic nephrotic syndrome in adults [1,2]. Two different forms of MN have been identified: a primary of idiopathic form (IMN), which is found in 80% of MN patients, and a secondary form associated with various malignancies, autoimmune diseases and some infections [1,2]. In the 1950s, Heymann and his associates developed an experimental animal model wherein rats developed severe proteinuria after active or passive immunization with certain antigenic fractions of proximal tubular brush border [4]. The histopathological features of renal involvement in this model were similar to those seen in human IMN and additional studies showed that antibodies bound to a membrane receptor expressed on rat renal podocytes, which was eventually identified as megalin [5]. In contrast to anti-megalin antibodies being capable of activating the complement pathway that leads to podocyte damage, antibodies of the IgG4 subclass that are characteristic of human MN, are believed to be ineffective activators of the classical pathway of complement [6,7]
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