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Multicenter Study
. 2010;12(3):R99.
doi: 10.1186/ar3029. Epub 2010 May 20.

Clinical and serological evaluation of a novel CENP-A peptide based ELISA

Affiliations
Multicenter Study

Clinical and serological evaluation of a novel CENP-A peptide based ELISA

Michael Mahler et al. Arthritis Res Ther. 2010.

Abstract

Introduction: Anti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA.

Methods: Sera collected from SSc patients (n=334) and various other diseases (n=619) and from healthy controls (n=175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies.

Results: The CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho=0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n=131) and various controls (n=134) was significantly better using the CENP-A as compared to CENP-B ELISA (P<0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P=0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P=0.0103), specific joint involvement (Jaccoud) (P=0.0006) and anti-phospholipid syndrome (P=0.0157) between ACA positive SLE patients and the entire SLE cohort were observed.

Conclusions: Anti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.

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Figures

Figure 1
Figure 1
Comparison between anti-CENP-A and anti-CENP-B reactivity. Correlation diagram is shown in a) Comparative receiver operating characteristic analysis for the discrimination between SSc patients (n = 131) and controls (n = 134) is shown in b). The area under the curve was significantly higher for CENP-A (0.81 vs. 0.47, P < 0.0001). Serum samples were selected that showed comparable results in CENP-A and CENP-B ELISA and diluted in Dilution Buffer to obtain a reactivity of approximately 1.0 OD. Increasing concentrations of CENP-A derived peptide and recombinant CENP-B were added to the diluted sera and incubated for two hours at room temperature. Specimens were then assayed in CENP-A and CENP-B ELISA. Inhibition was observed for anti-CENP-A reactivity with the CENP-A peptide c) and for anti-CENP-B reactivity with the recombinant CENP-B protein d), but not with the respective other antigen.
Figure 2
Figure 2
Receiver operating characteristics analysis. Receiver operating characteristics (ROC) analysis was performed using the data derived from all centres. Cut-off value of 1.5 RU is indicated by the arrows. ROC curve is shown in a) and ROC decision plot is shown in b) for the sensitivity and specificity.
Figure 3
Figure 3
Anti-CENP-A reactivity in different disease groups by comparative descriptive analysis. The prevalence and titre of anti-CENP-A measured by CENP-A peptide ELISA was significantly higher in SSc patients compared to controls. Except for rheumatoid arthritis, the anti-CENP-A titres were significantly higher in all disease groups compared to the healthy donors.

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