Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients’ and blood donors’ sera

Žigon, Polona; Lakota, Katja; Čučnik, Saša; Švec, Tinka; Ambrožič, Aleš; Sodin‐Šemrl, Snežna; Kveder, T

doi:10.3325/cmj.2011.52.694

Cited by 17 publications

(18 citation statements)

References 23 publications

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“…However, we detected increased levels of anti‐dsDNA antibodies using an in‐house ELISA and a commercial Luminex‐based assay (Figure E and Supplementary Figure 2F, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40535/abstract). In clinical practice, dsDNA ELISAs have shown low specificity and poor correlation with superior detection assays, such as CLIFT . We therefore tested Sle1 TLR‐9 −/− samples from Figures A–D with the CLIFT assay, and 6 of 8 serum samples showed positive kinetoplast staining, confirming the presence of anti‐dsDNA antibodies despite negative chromatin staining (Supplementary Figures 2A and B).…”

Section: Resultsmentioning

confidence: 90%

“…wiley.com/doi/10.1002/art.40535/abstract). In clinical practice, dsDNA ELISAs have shown low specificity and poor correlation with superior detection assays, such as CLIFT (35,36). We therefore tested Sle1TLR-9 À/À samples from Figures 2A-D with the CLIFT assay, and 6 of 8 serum samples showed positive kinetoplast staining, confirming the presence of anti-dsDNA antibodies despite negative chromatin staining ( Supplementary Figures 2A and B).…”

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confidence: 86%

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Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

Celhar

Yasuga

Lee

et al. 2018

Arthritis & Rheumatology

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ObjectiveToll‐like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR‐7 for disease development, genetic ablation of TLR‐9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR‐9 in the development of severe lupus in a mouse model.MethodsWe crossed Sle1 lupus‐prone mice with TLR‐9–deficient mice to generate Sle1 TLR‐9−/− mice. Mice ages 4.5–6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR‐7 protein expression. Mice ages 8–10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR‐7 expression.Results Sle1 TLR‐9−/− mice developed severe disease similar to TLR‐9–deficient MRL and Nba2 models. Sle1 TLR‐9−/− mouse B cells produced more class‐switched antibodies, and the autoantibody repertoire was skewed toward RNA‐containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1 TLR‐9−/− mouse renal DCs were more efficient at TLR‐7–dependent antigen presentation and expressed higher levels of TLR‐7 protein. Importantly, this increase in TLR‐7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction.ConclusionThe increase in TLR‐7–reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1 TLR9−/− mice.

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Section: Resultsmentioning

confidence: 90%

mentioning

confidence: 86%

Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

Celhar

Yasuga

Lee

et al. 2018

Arthritis & Rheumatology

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“…The most important causation of such result variability amongst the different assays is the affinity of autoantibodies towards the test antigen which is highly influenced by the reaction conditions. For example, some assays, as will be described shortly, favor the recovery of low-affinity antibodies such as ELISA, while others assays favor the recovery of high-affinity antibodies such as the Farr assay [27,97].…”

Section: Diagnostic Technologiesmentioning

confidence: 99%

“…The assay proceeds exactly as IFA where the Crithidia luciliae cells are fixed on a glass slide and a series of dilutions of the patient's serum is incubated with the cells and detection is mediated through the addition of fluorescently labeled anti-IgG antibodies. CLIFT has been described to be highly specific to SLE similar to the Farr assay [97]. However, DNA of Crithidia luciliae was described to have a bent conformation similar to nucleosomal DNA which can result in the recovery of only a subset of anti-DNA antibodies, and thus the assay was described to have a low sensitivity [27,97].…”

Section: Crithidia Luciliae Immunofluorescence Test (Clift)mentioning

confidence: 99%

“…CLIFT has been described to be highly specific to SLE similar to the Farr assay [97]. However, DNA of Crithidia luciliae was described to have a bent conformation similar to nucleosomal DNA which can result in the recovery of only a subset of anti-DNA antibodies, and thus the assay was described to have a low sensitivity [27,97].…”

Section: Crithidia Luciliae Immunofluorescence Test (Clift)mentioning

confidence: 99%

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SLE, An Overlooked Disease: Possibilities for Early Rescue by Early Diagnosis

Arafa¹,

Ahmed²

2018

Rapid Test - Advances in Design, Format and Diagnostic Applications

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Systemic lupus erythematosus (SLE) is a progressive autoimmune disease associated with widespread organ damage that can eventually cause death. Worldwide prevalence of SLE is difficult to report mainly due to difficulty in diagnosis as a result of its heterogeneous nature and nonspecific protean manifestations. Currently, circulating anti-DNA antibodies are the most specific diagnostic biomarkers for SLE where many detection assays are being employed in clinical practice. However, the diagnostic value of these techniques is challenged by the detection of only subpopulations of these antibodies with varying sensitivity and specificity. This is mainly attributed to differences in the antigen source and presentation and in the employed reaction conditions. This chapter will thoroughly discuss the technology, advantages, and limitations of each assay in addition to a special focus on the recently developed diagnostic technologies and novel biomarkers. Moreover, SLE will be presented as a disease model highlighting the importance of personalized medicine.

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

et al. 2018

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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.

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Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients’ and blood donors’ sera

Cited by 17 publications

References 23 publications

Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

SLE, An Overlooked Disease: Possibilities for Early Rescue by Early Diagnosis

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

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