Next, at room temperature, plates were washed thrice, blocked with 5% non-fat dry milk for 2 hours, incubated for 2 hours with serum diluted 1:100 in wash buffer with 5% milk, washed 4 times, incubated for 2 hours with goat anti-human IgG conjugated to horse radish peroxidase (Southern Biotech, Birmingham, USA) diluted 1:5000 in wash buffer with 5% milk, washed thrice, developed with 3,3,5,5-tetramethylbenzidine, and read at 450 nm with 540 nm correction. IgM binding to peptides were compared for CCP+RF+, CCP+RF?, CCP?RF+, and CCP?RF? rheumatoid Rabbit Polyclonal to OR5K1 arthritis versus controls (n=12). IgG-bound and endogenously citrullinated peptides were analyzed for amino acid patterns and predictors of intrinsic disorder, i.e. unstable three-dimensional structure. Binding to IgG-derived peptides was specifically evaluated. ELISA confirmed key results. Results: Broadly, CCP+RF+ subjects had high and CCP+RF? and CCP?RF+ subjects had modest NSC 23766 citrulline-specific IgG binding to array peptides (median z-scores: 3.02, 1.42, 0.75, respectively, p 0.0001). All rheumatoid arthritis groups had low homocitrulline-specific binding. CCP+RF+ subjects had moderate IgG binding to native peptides (median z-score 2.38, p 0.0001). The highest IgG binding was to citrulline-containing peptides, irrespective of protein identity, especially if citrulline was adjacent to glycine or serine, motifs also seen for endogenous citrullination in the rheumatoid joint. Highly bound peptides had multiple features predictive of disorder. IgG NSC 23766 from CCP+RF+ subjects targeted citrulline-containing IgG-derived peptides. Conclusion: Disordered antigens, which are frequently citrullinated, and common epitopes for ACPAs and RF are potentially unifying features for rheumatoid arthritis autoantibodies. In rheumatoid arthritis, autoantibodies are both pathologic (1C3) and diagnostic (4). Patients with rheumatoid arthritis produce a variety of anti-citrullinated protein antibodies (ACPAs) with overlapping reactivity (5C8) that underlie the diagnostic anti-cyclic citrullinated peptide antibody (CCP) tests. They also generate rheumatoid factor (RF), antibodies of any isotype that bind to the Fc portion of IgG, which is also used for diagnosis. Additionally, patients with rheumatoid arthritis make autoantibodies that target homocitrulline, called anti-homocitrullinated protein antibodies (AHCPAs) or anti-carbamylated protein antibodies (9). There appears to be some cross-reactivity between AHCPAs and ACPAs (7, 10C12), but this issue has not been completely resolved. Additionally, rheumatoid arthritis patients make autoantibodies against malondialdehyde-acetaldehyde adducted (13) and acetylated proteins (14), suggesting that autoantibodies in rheumatoid arthritis may primarily bind post-translationally modified proteins (15). However, native proteins also can be targeted in rheumatoid arthritis (16C18) and autoantibodies against post-translationally modified proteins often coexist with RF. Why these seemingly unrelated antigens are targeted in rheumatoid arthritis is a mystery. Although the majority of patients with rheumatoid arthritis generate ACPAs and RF, about 25% are seronegative for both CCP and RF (19). People with seronegative rheumatoid NSC 23766 arthritis may lack autoantibodies in general or common autoantibodies for this subset simply may not have been discovered yet. Additionally, some patients are seropositive for only RF or CCP. Little is known about autoantibody reactivity in single seropositive disease. However, an understanding of autoantibodies in these groups could shed light on the spectrum of disease in rheumatoid arthritis. Here we use a high density peptide array to evaluate autoantibodies against citrulline-containing, homocitrulline-containing and native peptides in seropositive and seronegative subjects in order to identify unifying and novel features of autoantibody reactivity in rheumatoid arthritis. MATERIALS AND METHODS Human Subjects: Human subjects research was carried out in compliance with the Helsinki Declaration and was approved by the University of Wisconsin Institutional Review Board. Serum from age- and sex-matched control and rheumatoid arthritis subjects were selected from the University of Wisconsin Rheumatology Biorepository first described in (20, 21). Briefly, rheumatoid arthritis subjects were identified by having 2+ outpatient visits with rheumatoid arthritis-associated ICD codes within 24 months (22) or one visit and a positive CCP test. Rheumatoid arthritis diagnosis was confirmed by manual review of the three most recent rheumatologist progress notes. Anti-CCP was assessed by generation II anti-CCP or anti-CCP3 ELISA (Inova, San Diego, USA) and RF was assessed by latex or polystyrene agglutination in the UW clinical lab. Rheumatoid arthritis subjects were included in the following groups if CCP and/or RF titers were negative or 2x the upper limit of normal: CCP+RF+, CCP-RF+, CCP+RF-, and CCP-RF-. Controls were excluded if they had any of the following as.