Future research is necessary to better define the clinical and serological characteristics of anti-Ro52Cpositive patients with SARD. Acknowledgments We thank Jana Langner (EUROIMMUN AG, Lubeck, Germany) for technical support. domain or domain association could increase their diagnostic specificity for any SARD. Methods Serum samples were obtained from 100 anti-Ro52 antibodyCpositive patients with SARDs and from 68 controls (50 healthy donors and 18 patients with other autoimmune or allergic diseases). A special line immunoassay was KLK3 created containing a full-length Ro52 antigen expressed in insect cells using the baculovirus PX 12 system, five recombinant Ro52 antigen fragments [Ro52-1, Ro52-2, Ro52-3, Ro52-4 (partly overlapping Ro52-1 and Ro52-2), and Ro52-5 (partly overlapping Ro52-2 and Ro52-3)], and two Ro52 peptides (176C196 aa and 200C239 aa), all expressed in amino acid, Zinc According to the literature, the CC domain is the main immunogenic region in patients with systemic lupus erythematosus (SLE), SjS, and IIM. The PX 12 C-terminal region of Ro52, containing the B30.2 domain, showed higher antibody titers only in patients with SjS [21, 22]. Several laboratory methods can be used to detect antibodies to the full Ro52 molecule (immunoenzymatic assay, line immunoassay, counterimmunoelectrophoresis, Western blot, chemiluminescence, and addressable laser bead immunoassay). Epitope recognition profiles have been analyzed only in anti-Ro52Cpositive mothers of children with congenital heart block to identify mothers at high risk for having affected children, and it has been suggested that a high ratio between antibody titers to 200C239 aa and 176C196 aa could be used as a marker of heart block risk [16, 17]. This approach stemmed from the observed dominant response to 200C239 aa in all mothers who gave birth to affected children, while antibodies against this peptide were significantly less frequent in mothers who gave birth to healthy children. Instead, sera from the latter mothers reacted mainly with epitopes contained within 176C196 aa of the Ro52 protein. The aim of our present study was to extend the examination of Ro52 epitope antibody profiles to patients with SARDs and to assess the prevalence of antibodies to the five Ro52 fragments and to Ro52 176C196-aa and Ro52 200C239-aa peptides. Furthermore, as antibodies against the entire Ro52 molecule are not disease-specific, we also aimed to evaluate if the antibodies against any single epitope or a combination of epitopes could have more disease specificity than antibodies against the full-length protein. Material and methods Patients Serum samples were obtained from 100 patients with SARDs and from 68 control subjects (50 healthy blood donors and 18 patients with other autoimmune or allergic diseases). The SARD group consisted of 23 patients with SLE, 34 with SjS, 7 with mixed connective tissue disease (MCTD), 27 with IIM, 2 with systemic sclerosis (SSc), and 7 with rheumatoid arthritis. SARD diagnoses were established according to internationally validated disease criteria. Patients with SARDs were selected on the basis of presence of anti-Ro52 antibodies in their sera. Because intermethod variability in measurement of anti-Ro52 antibodies is very high [23C25], Ro52 antibody presence was preliminarily confirmed using four different assays: a BioPlex 2200 antinuclear antibody (ANA) screen (Bio-Rad Laboratories, Hercules, CA, USA) and ANA Profile 3 EUROLine, Autoimmune Liver Disease Profile EUROLine, and Myositis Profile 3 EUROLine line immunoassays (EUROIMMUN, Luebeck, Germany). A serum sample was considered positive when anti-Ro52 antibodies were confirmed by at least three of these methods. Patient consent was not sought, owing to the retrospective nature of the study and the fact that it was carried out on leftover samples and because all analyses were performed blindly and patients records and information remained anonymous. Serological assays A special line immunoassay was PX 12 created that contained purified full-length Ro52 antigen expressed in insect cells using the baculovirus system and five recombinant Ro52 antigen fragments (Ro52-1CRo52-5, spanning aa residues 1C127, 125C268, 268C475, 57C180, and 181C320, respectively), according to UniProt accession number “type”:”entrez-protein”,”attrs”:”text”:”P19474″,”term_id”:”133250″,”term_text”:”P19474″P19474, produced in Mixed connective tissue disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sj?grens syndrome, amino acid, systemic autoimmune rheumatic disease Data are presented as number (%) Quantitative antibody values did not differ among the SARD cohorts for fragments 1, 3, 4, and 5 or for the 175C196-aa peptide, which provided negative results. In our analysis of the distribution of the intensity of the positive reactions, we found that antibody levels did not differ substantially for the full Ro52 molecule, but they did for fragment 2 (103??38 grayscale units) and for the 200C239-aa peptide (53??49), which displayed a higher antibody level in the MCTD and SjS cohorts (Fig.?2). However, no statistically significant combination of antibodies.