Clinical and serological evaluation of a novel CENP-A peptide based ELISA.
BACKGROUND
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.
Mahler
Michael
M
Dr. Fooke Laboratorien, Mainstrasse 85, 41469 Neuss, Germany.
Maes
Liesbeth
L
Blockmans
Daniel
D
Westhovens
Rene
R
Bossuyt
Xavier
X
Riemekasten
Gabriela
G
Schneider
Sandra
S
Hiepe
Falk
F
Swart
Andreas
A
Gürtler
Irmgard
I
Egerer
Karl
K
Fooke
Margrit
M
Fritzler
Marvin J
MJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D000076247:Centromere Protein A; D051936:Centromere Protein B / Q000276:immunology; D002868:Chromosomal Proteins, Non-Histone / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D012372:ROC Curve; D012595:Scleroderma, Systemic / Q000097:blood* / Q000276:immunology; D012680:Sensitivity and Specificity; D055815:Young Adult
D001323:Autoantibodies; D001324:Autoantigens; D015415:Biomarkers; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone