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Introduction The identification of specific autoantibodies can aid in the diagnosis, prognosis and management of systemic sclerosis (SSc). Commercial solid phase assays (including line blots and dot blots) are widely used for detection of SSc-specific and SSc-associated antibodies but may suffer from poor sensitivity or specificity for certain antibody specificities. Moreover, not all SSc-specific/associated antibodies can be detected by commercial assays, leaving a seronegative gap of ~10-15%. We aimed to characterize the antibodies that remained unidentified by commercial solid phase assays using immunoprecipitation combined with mass spectrometry (IP-MS). With IP-MS, antibodies can interact with epitopes of proteins or protein complexes, which can be directly identified through MS analysis without the need for an intermediary gel-based step.Material and Methods We analyzed samples from 135 patients diagnosed with SSc or SSc-myositis overlap syndrome with unknown autoantibodies from 8 cohorts [Cliniques Universitaires Saint-Luc (Belgium, n = 44), University of Calgary (Canada, n = 33), Chris Hani Baragwanath Hospital (South Africa, (n = 19), Centre Hospitalier Universitaire d’Angers (France, n = 11), Centre Hospitalier Lyon Sud (France, n = 11), Centre Hospitalier Universitaire de Lyon (France, n = 11), Nippon Medical School (Japan, n = 5) and University Medical Centre Ljubljana (Slovenia, n = 3) using IP-MS.Results SSc-specific/associated autoantibodies targeting Th/To (n = 19), RNA polymerase III (n = 8), Scl-70/topoisomerase 1 (n = 5), fibrillarin (n = 6) were identified in 37 patients. In 24 other patients SSc-myositis overlap syndrome-associated autoantibodies targeting PM/Scl (n=7), Ku (n=1), U1-RNP (n=9) and RuvBL1/2 (n=5, 1 patient also had anti-Th/To antibodies) were detected. In addition, autoantibodies against several rare SSc-associated autoantigens like the THO complex (n = 2), NVL (n = 3), RCC1 (n = 2), NOLC1 (n = 1) and NOP2 (n = 1) were identified. Two patients presented with myositis-specific autoantibodies (anti-Mi-2 and anti-Jo1). In three patients anti-mitochondrial autoantibodies were detected and in one patient anti-PCNA. Finally, in 5 other patients possible novel autoantibodies were identified. In total, an autoantibody could be identified in 56% of previously seronegative patients.Conclusions IP-MS identified autoantibodies in more than half of individuals with SSc or SSc-myositis overlap with previously unknown antibody specificity.Abstract P.040 Figure 1