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

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, 14-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.

A comparison of the chemiluminescence immunoassay and Crithidia luciliae immunofluorescence test in detecting anti-dsDNA antibodies and assessing the activity of systemic lupus erythematosus

“…11,[15][16][17] However, because of its high specificity, CLIFT is considered an effective method for diagnosing SLE and for distinguishing it from other autoimmune diseases. 18,19 Furthermore, because the results are semi-quantitative, it is difficult to follow the clinical course and predict SLE flares. By contrast, CLIA is a promising candidate method because of its good sensitivity, high specificity, and ability to provide quantitative results, thus enabling the tracking of level variations over time.…”

Anti-double stranded DNA antibodies: A rational diagnostic approach in limited-resource settings

“…However, Farr assay requires a special set-up for radioactive labeling of DNA and is also time-consuming, because of which its use has been declining in recent years, to be definitively abandoned in favor of other alternatives, such as that using intercalating fluorescent dsDNA dye, without the radioactive material and organic solvents [ 31 ]. Similarly, CLIFT may be considered as an alternative confirmatory test and its relevance seems increasingly defendable.…”

“…Similarly, CLIFT may be considered as an alternative confirmatory test and its relevance seems increasingly defendable. Indeed, despite its low sensitivity, globally ranging from 30% to 60% [ 19 , 25 , 32 , 33 ], but owing to its high specificity, CLIFT is considered useful in the diagnosis of SLE and to distinguish it from other diseases [ 17 , 31 , 34 ].…”

Comparative analysis of contemporary anti-double stranded DNA antibody assays for systemic lupus erythematosus

“…In further development of this approach, the polyethylene glycol (PEG) precipitation assay was created, a radioimmunoassay with modifications to also include the detection of low avidity anti-dsDNA antibodies ( 14 ). The next evolutionary step aimed at the elimination of radioisotope utilization and gave rise to the Farr fluorescent immunoassays [Farr-FIA ( 15 )] and the Crithidia luciliae immunofluorescent test (CLIFT). Finally, the trinity of anti-dsDNA assay methodologies was completed with the establishment of enzyme-linked immunosorbent assays (ELISA), which have evolved to be today’s most commonly used anti-dsDNA testing method ( 16 ).…”