Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Abstract

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3-10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11-16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-alpha) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-alpha peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-alpha and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-alpha peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-alpha peptide antibodies. These data indicate that anti-PM1-alpha antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-alpha ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.

Figure 1

Correlation diagrams of PM1-α, PM/Scl-75a, PM/Scl-75c and PM/Scl-100. A panel of sera tested previously for reactivity to recombinant polymyositis/scleroderma (PM/Scl) components (PM/Scl-75a, PM/Scl-75c and PM/Scl-100) was assayed for anti-PM1-α peptide reactivity in an ELISA [18]. Correlation diagrams are shown comparing the peptide ELISA with the recombinant proteins (a)–(c) for all sera (n = 81) and (b)–(f) for only the sera of PM/Scl patients (n = 36).

Figure 2

Receiver operating characteristic analysis of the PM1-α ELISA. Results obtained from three centers and based on 567 patients including polymyositis/scleroderma (PM/Scl) patients (n = 40), Scl patients (n = 205) and PM patients (n = 40) as well as other controls were used to calculate a receiver operating characteristic analysis (a) for all control samples and (b) for unrelated controls (without Scl and PM). The curve shows a clear discrimination between PM/Scl patient samples and various controls as emphasized by an area under the curve value of 0.901 (all controls) and 0.958 (unrelated controls). The differentiation between PM/Scl patients and controls was significantly improved when Scl patients and PM patients were excluded from the control group (b). SE, standard error.

Figure 3

Reactivity of polymyositis/scleroderma (PM/Scl) patients and controls in the PM1-α ELISA. Results obtained from three centers and based on 567 patients including PM/Scl patients (n = 40), Scl patients (n = 205) and PM patients (n = 40) as well as other controls were used to calculate comparative descriptive analysis. The diagram shows a significantly increased reactivity of the PM/Scl sera compared with the control groups. Comparative descriptives show vertical box-plots for each sample, side by side for comparison. The blue line series shows parametric statistics: diamond, mean and the requested confidence interval around the mean; notched line, requested parametric percentile range. The notched box and whiskers show non-parametric statistics: notched box, median, lower and upper quartiles, and confidence interval around the median; dotted line, connects the nearest observations within 1.5 interquartile ranges (IQR) of the lower and upper quartiles. + and ○, possible outliers – observations more than 1.5 IQR (near outliers) and more than 3.0 IQR (far outliers) from the quartiles. Vertical lines, requested nonparametric percentile range. SLE, systemic lupus erythematosus; HCV, hepatitis C virus; RA, rheumatoid arthritis.