Open Access Research article

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

Michael Mahler12, Liesbeth Maes3, Daniel Blockmans3, Rene Westhovens3, Xavier Bossuyt3, Gabriela Riemekasten4, Sandra Schneider4, Falk Hiepe4, Andreas Swart5, Irmgard Gürtler5, Karl Egerer6, Margrit Fooke12 and Marvin J Fritzler7*

Author Affiliations

1 Dr. Fooke Laboratorien, Mainstrasse 85, 41469 Neuss, Germany

2 Current address: Department of Immunopathology, INOVA Diagnostics, San Diego, CA 92131-1638, USA

3 Laboratory Medicine, General internal medicine, Rheumatology, University Hospitals Leuven, Binkomstraat 2 3210 Lubbeek, Belgium

4 Charité University Hospital, German Rheumatism Research Centre, a Leibniz institute, Dept Rheumatology and Clinical Immunology Charitéplatz 1, 10117 Berlin, Germany

5 Rheumatology Clinical Neuss, Neuss 41460, Germany

6 Interdisziplinäres Autoimmun-Speziallabor, Charité University hospital, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany

7 Faculty of Medicine, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, T2N 4N1, Canada

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Arthritis Research & Therapy 2010, 12:R99  doi:10.1186/ar3029

Published: 20 May 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.