Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Background/Purpose: Robust, certified, and traceable reference material for autoantibody testing is vital for the validity of results obtained in the clinical laboratory. International standards for qualitative and quantitative immunologic assays are traditionally based on large batches of serum/plasma obtained from a single individual. However, autoantibodies from one single individual may not appropriately represent the vast heterogeneity of autoantibodies observed in human species. Autoantibodies to the 70/75kDa
Methods: Serum samples with the characteristic DFS pattern at titer ≥1/640 were sequentially selected in the laboratory ANA practice and kept at -80oC. Validation of samples and pools included agreement on the characteristic ANA DFS pattern by 3 independent examiners and demonstration of the typical 75kDa band on western blot and moderate/strong anti-DFS70 reactivity in 2 independent methods [ELISA (cut-off>1.0) and chemiluminescent assay (CLIA; cut-off>20)]. A progressive pooling strategy included the formation of PENTA pools (5 validated samples), ICOSA pools (4 validated PENTA), SEMI-FINAL pools (3-5 validated ICOSA) and the FINAL POOL (8 validated SEMI-FINAL). The FINAL POOL was serially diluted in fetal bovine serum (FBS) to verify the linearity and increase the volume adjusting to moderate reactivity.
Results: The 760 validated samples (CLIA median 337.7U; 26.1–3,547.0) yielded 152 validated PENTA (CLIA: 420.6U; 60.4-3,518; ELISA: 5.5U; 2.8-6.8). The 152 PENTA yielded 38 validated ICOSA (CLIA: 407.2U; 77.5-1,823.3; ELISA: 5.5U; 3.7-6.7), 8 SEMI-FINAL pools (ELISA 5.2U; 3.8-6.2) and FINAL pool (ELISA 5.1U). Intermediary pools and the FINAL pool exhibited the characteristic DFS pattern and a clear-cut 75kDa band in WB. Serial dilution in FBS resulted in a linear decreasing reactivity in all methodological platforms.
Conclusion: This proof-of-concept study indicates that pooling samples from hundreds of individuals with high titer anti-DFS70 reactivity preserves the original reactivity of individual samples encompassed, as judged by the ANA, CLIA, ELISA, and WB assays. The present anti-DFS70 standard will integrate the panel of ANA standards of the Autoantibody Standardization Committee affiliated with the International Union of Immunology Societies (IUIS), to be distributed by the Centers for Disease Control. The extrapolation of this model to other autoantibody specificities must be validated by appropriate studies.
To cite this abstract in AMA style:Dellavance A, Baldo D, Andrade LEC. Establishment of an International Autoantibody Standard for Anti-DFS70/LEDGF Antibodies: Proof-of-Concept Study for a Novel Strategy for the Setting up of International Autoantibody Standards [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/establishment-of-an-international-autoantibody-standard-for-anti-dfs70ledgf-antibodies-proof-of-concept-study-for-a-novel-strategy-for-the-setting-up-of-international-autoantibody-standards/. Accessed December 3, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/establishment-of-an-international-autoantibody-standard-for-anti-dfs70ledgf-antibodies-proof-of-concept-study-for-a-novel-strategy-for-the-setting-up-of-international-autoantibody-standards/