Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus

Systemic lupus erythematosus is characterized by antibodies to a variety of intracellular self-antigens, such as dsDNA and Sm, and these serve as hallmarks in the diagnosis of systemic autoimmune diseases. Several studies have shown that SmD1 and SmD3 synthetic peptides represent highly functional antigens for autoantibody detection and thus for diagnostic applications. The present study analysed the technical and clinical accuracy of an anti-SmD1 (amino acids 83–119) and an anti-SmD3 (amino acids 108–122) ELISA for the detection of anti-Sm antibodies. Depending on the cut-off value of the SmD1 ELISA, we found a high degree of concordance between the two tests. At an optimized cut-off value of 100 units for SmD1 we found the same clinical sensitivity (12.5%) and specificity (100%) in a group of systemic lupus erythematosus patients (n = 48) and in controls (n = 99). The concordance at this cut-off value was 100% (P < 0.0001; χ2 = 127.61). Using a second panel of sera (n = 65) preselected based on positive anti-Sm results, we confirmed the high degree of concordance between the two assays. Using dsDNA-coated ELISA plates and biotinylated peptides we confirmed the high dsDNA binding properties for SmD1, which were significantly higher than the SmD3-derived peptide. However, no cross-linking of anti-dsDNA antibodies to SmD1 was observed after adding increasing amounts of dsDNA to anti-dsDNA positive, anti-SmD1 negative serum. We therefore conclude that the reported difference in the sensitivity is related to the different cut-off levels and not to the detection of anti-dsDNA antibodies bridged via dsDNA to the SmD1 peptide. Moreover, we found that a subpopulation of anti-Sm antibodies cross-reacted with SmD1 and SmD3. Taken together, the data indicate that both SmD peptide ELISAs represent accurate assays and may be used as important standards for the detection of anti-Sm antibodies

Evaluation of Different Immunoassays for the Detection of Anti- dsDNAAutoantibodies in Patients with Systemic Lupus Erythematosus (SLE)

Der Nachweis der Anti-dsDNA-Autoantikörper im Serum ist von hoher diagnostischer Relevanz beim Systemischen Lupus Erythematodes (SLE). Die eingesetzten Methoden zu ihrer Bestimmung weisen jedoch mitunter große qualitative Unterschiede auf. Aus diesem Grunde wurden vier verschiedene kommerzielle ELISA (Farrzyme, Bindazyme, Orgentec und Varelisa), der Farr- Radioimmunoassay (Farr-RIA), und der Crithidia luciliae- Immunfloureszenz-Test (CLIF) hinsichtlich ihrer diagnostischen Wertigkeit beim SLE im Vergleich zu Kontrollgruppen, bestehend aus Patienten mit anderen entzündlichen Erkrankungen untersucht. Eine hohe Spezifität für den SLE wiesen insbesondere der CLIF mit 99% und der Farr-RIA mit 88% auf, wobei der Farr-RIA auch noch eine hohe diagnostische Sensitivität von 85% hatte, während der CLIF deutlich weniger sensitiv war (68%). Die diagnostische Spezifität der vier kommerziellen ELISA-Tests, die zwischen 71 und 78% lag, war diesen beiden Tests merklich unterlegen. Die Sensitivität von 3 der 4 ELISA-Tests war jedoch vergleichbar hoch bis höher als im Farr-RIA. Der Farrzyme-ELISA hatte eine etwas höhere diagnostische Spezifität (78%), aber dafür auch eine niedrigere Sensitivität von 70% als die anderen ELISAs. Die mit dem Farrzyme-ELISA, Farr- RIA und CLIF gemessenen Anti-dsDNA-Antikörper-Spiegel waren mit einer Nierenbeteiligung und niedrigen Komplementspiegeln C3 und C4 assoziiert. Für diese 3 Tests, sowie 2 weitere ELISAs (Bindazyme und Orgentec) konnte ein Zusammenhang mit der Krankheitsaktivität gezeigt werden. Die Ergebnisse bestätigen, dass sich die verschiedenen Tests qualitativ unterscheiden. Für die Diagnose eines SLE sind insbesondere der CLIF und der Farr-RIA von großer Bedeutung. Positive Testergebnisse im CLIF und Farr-RIA finden sich fast ausschließlich bei SLEPatienten. Da in den Routine-Laboratorien hauptsächlich ELISAs zum Nachweis von AntidsDNA- Antikörpern eingesetzt werden, sollte bei unklarer Diagnose ein positives Testergebnis immer im CLIF und/oder Farr-RIA bestätigt werden. Die hochempfindlichen ELISAs können bei gesicherter SLE- Diagnose zum Monitoring der Krankheitsaktiviät eingesetzt werden.The detection of anti-dsDNA antibodies plays an outstanding role in the diagnosis of systemic lupus erythematosus (SLE). However, the quality of methods used for SLE diagnosis may differ greatly. For this reason, we compared the diagnostic value of four commercial ELISAs (Farrzyme, Bindazyme, Orgentec and Varelisa) with that of the Farr radioimmunoassay (Farr- RIA) and the Crithidia luciliae immunofluorescence test (CLIF). Each was used to test serum samples from SLE patients versus control groups of patients with other inflammatory diseases. CLIF and Farr-RIA proved to be highly specific for SLE (99% and 88%, respectively). The diagnostic sensitivity of Farr-RIA was high (85%), whereas CLIF was significantly less sensitive (68%). The diagnostic specificity of the four ELISAs was lower (71% to 78%). In contrast, the diagnostic sensitivity of 3 out of 4 ELISAs was comparable or higher than that of Farr-RIA. The Farrzyme ELISA had slightly greater diagnostic specificity (78%), but was less sensitive (70%) than other ELISAs. Anti-dsDNA antibody detection by Farrzyme ELISA, Farr-RIA and CLIF was associated with renal involvement and low levels of complement components C3 and C4. Anti-dsDNA antibodies measured by these threeassays and two ELISA methods (Bindazyme and Orgentec) were related to disease activity. These results confirm that there are notable quality differences between the studied antidsDNA antibody assays. CLIF and Farr-RIA are particularly valuable for SLE diagnosis. With CLIF and Farr-RIA, positive anti-dsDNA antibody results were obtained almost exclusively in SLE patients. Consequently, positive anti-dsDNA antibody results by the ELISA tests most commonly used in routine laboratories should be confirmed by CLIF and/or Farr-RIA to establish the definitive diagnosis of SLE. The ELISA methods are highly sensitive and can be used to monitor disease activity in patients with an already confirmed diagnosis of SLE

Receiver-operating characteristic analysis of two SmD peptide-based anti-Sm antibody assays

<p><b>Copyright information:</b></p><p>Taken from "Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus"</p><p>http://arthritis-research.com/content/9/4/R68</p><p>Arthritis Research & Therapy 2007;9(4):R68-R68.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC2206372.</p><p></p> The results of this comparative study were used to generate receiver-operating characteristic curves. The discrimination between systemic lupus erythematosus patient samples and control samples was similar for both SmD immunoassays

The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus

Objectives To investigate whether bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, induces clinically relevant plasma cell (PC) depletion in patients with active, refractory systemic lupus erythematosus (SLE). Methods Twelve patients received a median of two (range 1–4) 21-day cycles of intravenous bortezomib (1.3 mg/m2) with the coadministration of dexamethasone (20 mg) for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti–double-stranded DNA (anti-dsDNA) and vaccine-induced protective antibodies were monitored. Flow cytometry was performed to analyse peripheral blood B-cells, PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN) activity. Results Upon proteasome inhibition, disease activity significantly declined and remained stable for 6 months on maintenance therapies. Nineteen treatment-emergent adverse events occurred and, although mostly mild to moderate, resulted in treatment discontinuation in seven patients. Serum antibody levels significantly declined, with greater reductions in anti-dsDNA (∼60%) than vaccine-induced protective antibody titres (∼30%). Bortezomib significantly reduced the numbers of peripheral blood and bone marrow PCs (∼50%), but their numbers increased between cycles. Siglec-1 expression on monocytes significantly declined. Conclusions These findings identify proteasome inhibitors as a putative therapeutic option for patients with refractory SLE by targeting PCs and type-I IFN activity, but our results must be confirmed in controlled trials