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Autoantibodies against dsDNA measured with nonradioactive Farr assay—an alternative for routine laboratories

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Abstract

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

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Authors and Affiliations

  1. Department of Rheumatology, University Medical Centre Ljubljana, Vodnikova 62, 1000, Ljubljana, Slovenia

    K. Lakota, T. Švec, T. Kveder, S. Sodin-Šemrl, P. Žigon, A. Ambrožič, M. Ogrič, S. Markez, B. Božič, M. Tomšič & S. Čučnik

  2. Faculty of Mathematics, Natural Science and Information Technology, University of Primorska, Koper, Slovenia

    K. Lakota & S. Sodin-Šemrl

  3. Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

    B. Božič & S. Čučnik

  4. Medical Faculty, University of Ljubljana, Ljubljana, Slovenia

    M. Tomšič

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  1. K. Lakota
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Correspondence to K. Lakota.

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Disclosure

The study is part of an approved national patent no. WO/2015/119582 (Fluorometric immunoassay for detection of anti-dsDNA antibodies, patent: Publication Date: 13.08.2015) and international patent pending (#PCT/SI2015/000003, International Filing Date: 03.02.2015. Geneva: World Intellectual Property Indicators, 2015).

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Rheumatology in Slovenia: Clinical practice and translational research

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Lakota, K., Švec, T., Kveder, T. et al. Autoantibodies against dsDNA measured with nonradioactive Farr assay—an alternative for routine laboratories. Clin Rheumatol 38, 353–359 (2019). https://doi.org/10.1007/s10067-018-4271-3

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  • DOI: https://doi.org/10.1007/s10067-018-4271-3

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