Abstract / Summary
Objectives Emerging evidence supports the presence of autoantibodies targeting interleukin-1 receptor antagonist (IL-1Ra), a regulator of IL-1 signaling, in adult-onset Still’s disease (AOSD). However, existing data are limited by small cohorts and potential confounding due to prior exposure to IL-1 inhibitors. This study aimed to develop a simple and reproducible indirect ELISA for detecting anti-IL-1Ra antibodies and to assess their frequency and immunological features in IL-1 inhibitor-naïve AOSD patients. Methods Serum samples from IL-1 inhibitor-naïve AOSD patients (n=46) and healthy controls (n=26) were analyzed using an indirect ELISA based on recombinant IL-1Ra (anakinra) as antigen. Specificity was confirmed by dot blot. Anti-IL-1Ra IgG were affinity-purified from a high-titer patient and characterized by ELISA and SDS-PAGE. Epitope mapping was performed using phage display peptide libraries, followed by sequence alignment and structural mapping onto native IL-1Ra. Results Anti-IL-1Ra antibody levels were significantly higher in AOSD patients than in healthy controls (p=0.003), with a greater proportion showing high titers (p<0.001). Levels did not differ between active and inactive AOSD patients. Purified antibodies displayed specific dose-dependent binding to IL-1Ra. Phage display identified two peptide motifs corresponding to linear epitopes mapped to surface-exposed regions of the native protein, indicating recognition of unmodified IL-1Ra domains. Conclusions Anti-IL-1Ra antibodies are detectable in IL-1 inhibitor-naïve AOSD patients using a standardized ELISA. The identification of distinct linear epitopes suggests a heterogeneous autoimmune response targeting IL-1Ra and may support a role for adaptive immunity within the Still’s disease spectrum. Further studies are needed to define their functional and clinical relevance.