Session Type: Abstract Submissions (ACR)
Background/Purpose: Scleroderma (systemic sclerosis or SSc) has a highly variable clinical presentation and course resulting in difficulties for disease management. When SSc is suspected, autoantibodies (AAB) are tested. The most frequently observed AABs, anti-topoisomerase I (anti-Scl70), anti-centromere (ACA) and anti-RNA polymerase III (ARA) are associated with different subtypes of SSc and with specific organ involvements and disease severity. Those biomarkers have recently been included in the new SSc classification criteria, but are only present in about 60-70% of SSc patients. Our aim is to identify additional highly frequent SSc-associated autoantigens using a high-throughput Luminex bead-based profiling platform as well as the development of a complementary ELISA-based autoantibody assay kit for enabling validation of the novel panel of SSc antigens to illustrate their clinical utility.
Methods: A systematic and undirected approach was undertaken to screen for autoantibodies in human serum samples of 100 individuals with SSc and related overlap syndromes using 7000 recombinant protein targets. Active and passive control groups comprised healthy controls and serum samples of other systemic autoimmune diseases including systemic lupus erythematosus (SLE), and early rheumatoid arthritis (RA). The frequency of autoantibodies for a particular antigen was determined by applying the mean value of the signal intensity of the healthy control cohort plus 2 standard deviations (SD) as a cut-off. Then, the frequency of 116 candidate antigens in 1100 healthy controls and 90 SSc samples was assessed using a connective tissue disease array. Afterwards, ELISAs were developed for 7 antigens with high frequency in SSc or higher frequency in SSc subtypes. The performance of ELISAs was analyzed in comparison with the Luminex multiplex system in additional 150 SSc samples.
Results: Sera of 100 SSc patients with limited or diffuse SSc or overlap syndromes were tested for established and novel autoantigens. 30% of SSc patients were previously tested negative for ACA and anti-Scl70. The frequency of established and novel autoantibodies in the test cohort ranged from 40% to 10% in descending order: CENPB: 40%, Scl70: 36%, TRIM21 (Ro52): 27%, antigen 1: 28%, antigen 2: 27%, antigen 3: 17%, antigen 4: 15%, antigen 5: 13%, antigen 6: 11% and antigen 7: 4%. While autoantibodies to antigens 1-4 were more frequently observed in limited SSc, autoantibodies to antigens 5-7 were more abundant in diffuse SSc. Autoantibody reactivity above the cut-off level was identified in 50% of the previously negative-tested SSc patients. ELISA and Luminex showed good qualitative agreement. The genes encoding for these proteins were found being enriched in pathways of histone modifications and chromatin remodeling suggesting their involvement in epigenetic processes.
Conclusion: Using a combination of Luminex bead-arrays for high-throughput autoantibody profiling and complementary ELISA development provides an alternative route to discover and verify novel SSc-associated autoantibodies. By measuring 7 antigens the number of autoantibody positive SSc patients increased from 68% to 84%.
H. D. Zucht,
P. D. M. Schneider,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-novel-scleroderma-associated-antigens-and-development-of-an-autoantibody-assay-panel-enabling-their-subsequent-validation/