Proteomic Identification Of Novel Autoantibody Targets In Rheumatoid Arthritis And Diagnostic Performance Of Tgf-Beta-1-Induced Protein And Serum Amyloid A Autoantibodies

Objective: The principal diagnostic test for rheumatoid arthritis (RA) relies on the detection of anti-cyclic citrullinated peptide (CCP) antibodies. The CCP test exhibits moderate sensitivity and specificity but does not adequately detect a high proportion of cases or effectively distinguish RA from related conditions such as systemic lupus erythematosus (SLE). To discover improved diagnostic biomarkers for RA, we performed a comprehensive proteomic analysis of antibody-bound antigens present in RA, SLE, and healthy control (HC) sera and identified proteins associated with transforming growth factor-beta-1 (TGF-beta-1) signaling and assessed the sensitivity and specificity of antibodies against these antigens in comparison to CCP. Methods: Total antibodies were immunoprecipitated from pooled sera of HC individuals (n=3) and patients with clinically diagnosed RA (n=3) or SLE (n=3). Antibody-bound antigens were identified using multidimensional protein identification technology (MudPIT) shotgun proteomics. Candidate antigens associated with TGF-beta-1 signaling were selected for evaluation, and corresponding serum autoantibodies were assessed for their ability to classify RA using a POC lateral-flow immunochromatographic format. Results: Proteins present in antibody immunoprecipitations included TGF-beta-1-induced protein (TGFBI), thrombospondin 1 (TSP1), serum amyloid A1 (SAA), and fibronectin (FN). POC assays using these candidates, as well as TGF-beta-1 itself, as capture antigens were used to detect corresponding serum autoantibodies in 107 subjects with clinically diagnosed RA, 42 subjects with other rheumatic diseases (non-RA), and 121 HC subjects. Receiver operating characteristic curves were constructed using RA and HC groups to assess diagnostic performance, and optimal cutpoints identified using the Youden Index (maximizing the sum of sensitivity and specificity). CCP test results were determined using previously established thresholds. Using an analysis et of 169 subjects in the HC and RA groups who had values for TGFBI, SAA, and CCP, TGFBI autoantibodies achieved a sensitivity of 82%, specificity of 98.8%, positive likelihood ratio (PLR) of 65.6, and NLR of 0.2. SAA autoantibodies achieved a sensitivity of 92.1%, specificity of 85%, PLR of 6.1, and NLR of 0.1. Both outperformed CCP (sensitivity of 68.5%, specificity of 95%, PLR of 13.7, and NLR of 0.3). Using an analysis set of 120 subjects in the RA and non-RA groups who had values for TGFBI, SAA, and CCP, TGFBI and SAA autoantibodies exhibited sensitivities and specificities of 82 and 92.1% and 74.2 and 45.2%, respectively, compared to 68.5 and 74.2% for CCP. Autoantibodies to TGF-beta-1, TSP1, and FN exhibited only modest diagnostic performance. Conclusion: RA sera contain autoantibodies targeting multiple proteins associated with the TGF-beta-1 signaling pathway that is implicated in inflammatory processes in RA. Among the candidates tested, TGFBI and SAA autoantibodies exhibited the highest diagnostic performance in a POC lateral-flow format, supporting their potential utility as biomarkers for RA diagnosis. These findings also support further investigation of the TGF-beta-1 signaling pathway as a potential therapeutic target in RA.