Comparative qualitative and quantitative analysis of scleroderma (systemic sclerosis) serologic immunoassays
Introduction
Systemic sclerosis (SSc) is a chronic multisystemic disorder of unknown etiology characterized by vascular damage and by deposition of collagen in various tissues [1]. SSc-specific autoantibody profiles to a variety of cellular components [2], [3] are strongly associated with distinct clinical phenotypes making serologic testing of great diagnostic aid [4]. The most prominent ones are anti-topoisomerase I (Topo I), anti-centromere (ACA) and anti-nucleolar antibodies [5]. Anti-Topo I antibodies occur in patients with diffuse systemic sclerosis (dSSc) and have been associated with pulmonary involvement and digital ulcers [6] whereas anticentromere antibodies are mainly detected in patients with limited systemic sclerosis (lSSc) [2], [7], [8].
ACA recognize the centromere proteins A, B, and C (CENP-A, 17 kD; CENP-B, 80 kD; CENP-C, 140 kD) as shown by Immunoblots using nuclear constituents as antigen source [9], [10]. Topo I is a nuclear enzyme responsible for interconverting different topological forms of DNA [11]. Anti-Topo I antibodies can be detected either by Immunoblots of purified HeLa cell chromosome or nuclei and/or by Immunoblots on fragments of recombinant topoisomerase I [12], [13]. It has also been proved that ELISA with purified topoisomerase I as antigen source can provide a useful diagnostic and prognostic tool [14] and it is more sensitive than gel diffusion and more specific than Immunoblot but anti-Topo I detection improvement is still investigated [13], [15].
Several antibodies targeting nucleolar antigens are found most frequently in patients suffering from SSc and overlap syndromes [16], [17], [18]. Antinucleolar antibodies (ANoA) are almost exclusively detected in SSc patients' sera by immunoprecipitation assays including antibodies against the U3 small nucleolar RNP (U3snoRNP) complex or fibrillarin [19], [20], [21], [22], [23], the PM–Scl complex [24], [25], the nucleolar 7–2 RNA (Th/To ribonucleoprotein) [23], [26], [27], the upstream binding factor NOR-90 [28] and the nucleolar phosphoprotein B23 [29].
A number of studies have demonstrated the presence of autoantibodies against the three classes of RNA polymerases detected by immunoprecipitation assays using 35S-methionine radiolabelled extracts and ELISA testing [30], [31], [32], [33], [34].
Furthermore, additional autoantibody specificities such as anti-histone antibodies (AHA) detected by ELISA and Immunoblot [35] in SSc patients' sera, autoantibodies to pyruvate dehydrogenase complex also detected by ELISA and Immunoblot [36] and antiubiquitin antibodies detected by ELISA [37] have been described. The prevalence of anti-U1RNA antibodies [38] and the presence of anti-U5 snRNPs as possible scleroderma-associated serologic marker [39] have been recently reported.
The heterogeneity of autoantibody populations appearing in SSc, and the difficulty of their detection by routine assays such as Indirect Immunofluorescence (IIF) or Counterimmunoelectrophoresis (CIE), raise the necessity of developing sensitive and specific techniques for their identification. To study the variety of autoantibody specificities appearing in SSc patients' sera, four methods have been applied including IIF, CIE, Immunoblot on nuclear extract and different subfractions of nuclei as well as RNA precipitation; the sensitivity and specificity of these assays were assessed. Selected sera from well characterized patients covering all the disease variants (dSSc, lSSc, overlap syndrome) and their related clinical characteristics were used in the present study.
Section snippets
Sera
Serum samples from 150 patients with SSc were selected for this study. Patients were outpatients from the rheumatology clinic of Ioannina University Hospital and the rheumatology clinic of Laikon University Hospital of Athens and included 84 patients with lSSc, 54 with dSSc and 12 with overlap syndrome. The diagnoses were based on the Le Roy criteria [40]. Serum samples from 20 blood donors were included in the study. Reference sera for anti-U3RNP and anti-Th/To antibodies were kindly provided
Immunoblot with nuclear HeLa cell extract (IB-nuclear)
Nuclear HeLa cell extract was prepared as previously described [42]. Briefly 2.5 × 107 cells/ml were suspended in buffer A containing 10 mM Tris–HCl, 100 mM NaCl, 2.5 mM MgCl2, 0.5% v/v Triton X-100 and 2 μg/ml pepstatin, leupeptin, aprotinin. After homogenization with a type-S pestle and centrifugation at 4000 rpm for 10 min at 4 °C, the nuclei were separated, resuspended in buffer A and then sonicated on ice twice for 5 s while the temperature was maintained below 4 °C. The sonicate was then layered on a
Definition of sensitivity and specificity/statistical analysis
Sensitivity of a method is defined as the percentage (%) of the number of positive results found by this method towards the real positive results obtained by the reference method.
Specificity of a method is defined as the percentage (%) of the number of negative results found by this method towards the real negative results obtained by the reference method.
Clinical characteristics of the selective SSc patient population studied
One hundred fifty patients with SSc variants, either lSSc (n = 84), dSSc (n = 54) or overlap syndrome (n = 12) were evaluated for their autoantidody profile. As shown in Table 1, the above patients were completely representative of a severe Scleroderma patients' cohort. A large proportion of these patients have noticed digital ulcers, telangiectasias and esophageal dysmotility associated symptoms such as dysphagia or retrosternal burning sensation. A small proportion of lSSc patients gradually
Discussion
Immunoblot techniques have been extensively used to detect autoantibodies to nuclear and cytoplasmic antigens associated with rheumatic diseases, and serologic as well as clinical correlations have been described [55]. In the present study we were able to identify various autoantibody specificities relatively specific for SSc, such as anti-Topo I, ACA, anti-fibrillarin/U3RNP, and other specificities not directly correlated with the disease, by means of Immunoblot using different crude extracts
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