Antiphospholipase A₂ receptor autoantibodies: a comparison of three different immunoassays for the diagnosis of idiopathic membranous nephropathy

Abstract

Background: The recent identification of circulating autoantibodies directed towards the M-type phospholipase A2 receptor (PLA2R) has been a major advancement in the serological diagnosis of idiopathic membranous nephropathy (IMN), a common cause of nephrotic syndrome in adults. The goal of this study was to compare the performance characteristics of two commercial assays as well as the first addressable laser bead immunoassay (ALBIA) developed for the detection of anti-PLA2R antibodies.

Methods: Serum samples of 157 IMN patients and 142 controls were studied. Samples were tested by a cell based immunofluorescence assay (CBA-IFA, Euroimmun, Germany), by ELISA (Euroimmun), and by a novel ALBIA employing an in vivo expressed recombinant human PLA2R.

Results: Overall, the three assays showed significant qualitative and quantitative correlation. As revealed by receiver operating characteristic analysis, the ALBIA correlated better with the CBA-IFA than the ELISA (P = 0.0003). The clinical sensitivities/specificities for IMN were 60.0% (51.0-68.5%)/98.6% (95.0-99.8%) and 56.2% (47.2-64.8%)/100.0% (97.4-100.0%) for ALBIA and CBA-IFA, respectively.

Conclusion: The ALBIA represents a promising assay for the detection of anti-PLA2R antibodies showing similar performance to the CBA-IFA and the advantage of ease of use and suitability for high throughput, rapid turnaround times, and multiplexing.

Figure 1

Correlation between different diagnostic immunoassays for the detection of anti-PLA₂R antibodies. In our cohort of 157 IMN patients and 41 nephrotic disease control patients, a total of 83 samples were positive and 82 were negative by all three methods, a concordance of 77.6% for all three methods. Overall qualitative agreements were 85.9% for CBA-IFA versus ELISA, 96.5% for CBA-IFA versus ALBIA, and 83.3% for ELISA versus ALBIA.

Figure 2

Comparative receiver operating characteristic (ROC) analysis (CBA-IFA positive versus negative samples). The ALBIA and the EUROIMMUN ELISA were compared to the EUROIMMUN CBA-IFA. The EUROIMMUN CBA-IFA was the first commercially available immunoassay for anti-PLA₂R and therefore was used to define the outcome (anti-PLA₂R positive versus anti-PLA₂R negative). With an area under the curve (AUC) of 0.99 (95% CI: 0.99 to 1.00), the ALBIA performed similar to the CBA-IFA assay. The ELISA reached an AUC of 0.94 (95% CI: 0.91 to 0.97). Cut-off values are indicated by the arrows. The ELISA was also analyzed with an alternative (borderline) cut-off of 14 units.

Figure 3

Comparative analysis (IMN versus controls). ALBIA and the CBA-IFA were compared to the diagnosis of the subjects tested. (a) With an area under the curve (AUC) of 0.78 (95% CI: 0.74–0.82) for CBA-IFA and of 0.84 (95% CI: 0.78–0.89) for ALBIA, both assays showed similar discrimination between IMN and controls. Cut-off values are indicated by the arrows. (b) Comparative descriptive analysis shows the prevalence of anti-PLA₂R antibodies in different cohorts measured by ALBIA.