Anti-dsDNA-NcX ELISA: dsDNA-loaded nucleosomes improve diagnosis and monitoring of disease activity in systemic lupus erythematosus

Abstract

Introduction: The objective of this study was to compare the clinical usefulness of the new anti-double-stranded DNA nucleosome-complexed enzyme-linked immunosorbent assay (Anti-dsDNA-NcX ELISA), which is based on dsDNA-loaded nucleosomes as antigens, with established test systems based on dsDNA or nucleosomes alone for systemic lupus erythematosus (SLE) diagnostics and determination of disease activity.

Methods: Sera from a cohort of 964 individuals comprising 207 SLE patients, 357 disease controls and 400 healthy donors were investigated using the Anti-dsDNA-NcX ELISA, Farr assay, Anti-dsDNA ELISA, Anti-nucleosome ELISA and Crithidia luciliae immunofluorescence (CLIF) assay, all of which are tests available from EUROIMMUN Medizinische Labordiagnostika AG (Lübeck, Germany). Receiver operating characteristic curve analyses were performed to compare the sensitivity and specificity of each assay. The test results yielded by these assays in a group of 165 fully characterized SLE patients were compared with the corresponding medical records.

Results: The Anti-dsDNA-NcX ELISA was found to have a sensitivity of 60.9% and a specificity of 98.9% in all 964 individuals at the manufacturer's cutoff of 100 U/ml. At a comparable specificity of 99%, the sensitivity amounted to 59.9% for the Anti-dsDNA-NcX ELISA, 54.1% for the Farr assay, 53.6% for the antinucleosome ELISA and 35.8% for the anti-dsDNA ELISA. The CLIF assay had a sensitivity of 28.0% and a specificity of 98.2%. The Anti-dsDNA-NcX ELISA correlated mostly with global disease activity in a cross-sectional analysis. In a longitudinal analysis of 20 patients with 69 patient visits, changes in Anti-dsDNA-NcX ELISA and antinucleosome ELISA results correlated highly with changes in disease activity over time.

Conclusions: The use of dsDNA-complexed nucleosomes as antigens in ELISA leads to optimized determination of diagnosis and disease activity in SLE patients and is available for clinical practice.

Figure 1

Right-positive and false-positive test results of anti-double-stranded DNA nucleosome-complexed enzyme-linked immunosorbent assay (Anti-dsDNA-NcX ELISA). (A) Scatterplot showing Anti-dsDNA-NcX ELISA immunoglobulin G results in 964 different sera. Dotted line represents the manufacturer's threshold (100 IU/ml). Values >800 IU/ml were set to 800 IU/ml for a clearer arrangement of the figure. SLE, systemic lupus erythematosus; SSc, systemic sclerosis; SS, Sjögren's syndrome; RA, rheumatoid arthritis; ND, normal donors; (B) Table showing all non-SLE patients who tested positive in the Anti-dsDNA-NcX ELISA listed with the test results of all measured assays and clinically relevant findings. Positive test results according to a comparable specificity of 98.9% are marked in bold. Nuc, anti-dsDNA-nucleosome ELISA; Farr, radioimmunoassay; CLIF, Crithidia luciliae immunofluorescence assay; ANA, antinuclear antibodies; C3, complement component 3.

Figure 2

Comparison of patient sera that tested positive in the anti-double-stranded DNA nucleosome-complexed enzyme-linked immunosorbent assay (Anti-dsDNA-NcX ELISA) and other investigated test systems shown in Venn diagrams. (A) Absolute numbers of patient sera that tested positive in the Anti-dsDNA-NcX ELISA (dsDNA-NcX), the anti-dsDNA ELISA (dsDNA) and the anti-dsDNA nucleosome ELISA (Nuc) and their combined intersections are shown. (B) The same analysis as in Figure 2A is shown, including different detection techniques using Anti-dsDNA-NcX ELISA, Farr assay (radioimmunoassay) and Crithidia luciliae immunofluorescence (CLIF) assay is presented. Cutoffs for all positive test results were read out of receiver operating characteristic curve analysis at a specificity of 98.15% because this specificity was calculated for the CLIF assay.

Figure 3

Changes in disease activity versus changes of six defined laboratory parameters over time. All results are based on a total of 69 patient visits of 20 different systemic lupus erythematosus patients. Delta values were calculated by subtracting values for a defined parameter from an actual visit from a defined parameter from the last visit (for example, ΔC3 = C3_{(visit n + 1)} - C3_{(visit n)}. Only changes in Anti-dsDNA-NcX and antinucleosome ELISA results correlated significantly with changes in modified Systemic Lupus Erythematosus Disease Activity Index 2000 (mSLEDAI 2000 [25]) score over time. The mSLEDAI items for double-stranded DNA (dsDNA) and complement components C3 and C4 were excluded to avoid bias. Linear regression analysis was used to calculate significance.