Autoantibodies to double-stranded DNA--intermethod comparison between four commercial immunoassays and a research biosensor-based device.

Patients with systemic lupus erythematosus (SLE) often develop a wide variety of serological manifestations including the presence of antibodies to double-stranded DNA (anti-dsDNA). Positivity for anti-dsDNA constitutes one of the laboratory criteria for the diagnosis of SLE and is therefore clinically relevant. We analyzed the diagnostic accuracies of four commercial anti-dsDNA immunoassays and compared the results with a recently established surface plasmon resonance (SPR) biosensor chip with covalently chip-immobilized dsDNA. The anti-dsDNA measurements were performed retrospectively in 50 patients with clinically proven SLE, 39 patients with other autoimmunopathies and 20 healthy controls. Data were evaluated by Receiver-Operator Characteristic (ROC) analysis, with special regard to SLE patients suffering from lupus nephritis. The ROC analyses for the four immunoassays and the SPR biosensor resulted in the following area-under-the-curve (AUC) and diagnostic efficiency (DE) values in descending order: Bindazyme AUC, 0.89; DE, 0.88; ELiA AUC, 0.89; DE, 0.86; SPR biosensor AUC, 0.82; DE, 0.80; Farrzyme AUC, 0.77; DE, 0.77; Farr AUC, 0.77; DE, 0.70. When considering the 22 nephritis SLE patients the following AUC were observed: Bindazyme 0.98; EliA 0.95; SPR biosensor 0.93; Farr 0.89; Farrzyme 0.88. Although various methodologies for the determination of anti-dsDNA were compared, the overall diagnostic accuracy was found satisfactory in all immunoassays. Best data were found for the Bindazyme assay. We referenced the measurements to our in-house SPR biosensor device which showed good AUC and DE values. When optimized, this technique, allowing to monitor antigen/ antibody interactions in real-time, may add a new analytical quality to the existing methods, potentially beneficial in diagnosis and clinical monitoring of SLE.

Autoantibodies to prothrombin and phosphatidylserine/prothrombin-complexes: do they contribute to the serodiagnosis of primary and secondary anti-phospholipid syndrome?

The diagnostic and clinical relevance of Ab to pure and phosphatidylserine-complexed prothrombin for primary and secondary APS was investigated in a total of 357 patients with (n = 169) and without (n = 188) connective tissue diseases. The overall frequency of anti-prothrombin Ab in sAPS, pAPS and patients without APS-related symptoms were found to be 50.0, 37.5 and 22.0%, respectively. From a total of 72 anti-prothrombin-positive samples, 12.5% were specific for pure prothrombin, 31.9% for phosphatidylserine/prothrombin-complexes and 55.6% recognized both antigenic forms. The simultaneous occurrence of other anti-phospholipid Ab was observed in 84% of all sera. Both types of anti-prothrombin Ab are significantly associated with lupus anticoagulant activity, but only Ab to pure prothrombin display such a relationship to clinical manifestations of APS. Based on these results, it cannot be recommended at present to include anti-prothrombin assays in the routine procedure for the serodiagnosis of APS. However, patients negative for lupus anticoagulant and typical APS-related anti-phospholipid Ab should be tested for anti-prothrombin reactivity, favoring, mainly due to its higher specificity, the ELISA containing pure prothrombin as antigen.

Reactivity profiles of autoantibodies to different phospholipids and the phospholipid-binding protein beta2-glycoprotein I in patients with clinical symptoms related to thromboembolic and/or vasculopathic events with or without connective tissue diseases.

To study the antigenic and epitope specificities of anti-phospholipid Ab in detail, we investigated 177 patients without (62 with APS-related systemic clinical symptoms, 115 with microangiopathies) and 164 patients with connective tissue diseases (CTD). Ab associated with primary APS (pAPS) seem to show a restricted specificity (phospholipid/beta2-GPI-complexes), whereas those in secondary APS (sAPS) react additionaly with pure beta2-GPI. Simultaneously, beta2-GPI-independent Ab were also frequently present in both conditions (50% of all Ab-positive sera). In CTD patients, the reactivity profile "pure beta2-GPI + phospholipid/beta2-GPI-complexes" is significantly associated with clinically manifest sAPS. Comparing cardiolipin and phosphatidylserine as antigenic target, the overall concordance (crossreactivity?) between both assays was lower than expected (52%), being highest in pAPS (87%) and sAPS (65%). Based on these results, a two-step procedure for reliable serological diagnosis of APS could be recommended: Ab-screening using a mix of phospholipids complexed with beta2-GPI (sensitivity > 90% for Ab concentrations above 20 U/ml) followed by an assay allowing the simultaneous detection of all relevant antigenic and epitope specificities.

Reactivities to the Sm autoantigenic complex and the synthetic SmD1-aa83-119 peptide in systemic lupus erythematosus and other autoimmune diseases.

The Sm antigenic complex is, besides dsDNA, the most important and specific autoimmune target in systemic lupus erythematosus (SLE). The population of anti-Sm Ab elicited is very heterogeneous in terms of epitope specificity resulting in a strong assay dependent detectability. Based on the description of a new autoantigenic target, the SmD1-aa83-119 peptide, we analysed 50 healthy persons and 205 patients with different autoimmune and other disorders with regard to their anti-Sm reactivities using different assays. The prevalence of anti-SmD1 peptide Ab and anti-Sm Ab in SLE was 36.0 (40/111) and 9.9% (11/111), respectively. The respective values obtained for non-SLE patients were 2.8 (4/144) and 5.3% (5/94). In SLE, anti-SmD1 peptide Ab are positively correlated to disease activity, nephritis and anti-dsDNA Ab. The association between reactivities of SLE samples in the traditional anti-Sm and the anti-SmD1 peptide ELISA was found to be 63.6%, contrasting markedly with the situation in non-SLE patients (no double-positive sera). SLE samples with an anti-Sm response restricted to the SmD1 peptide are completely negative in immunoblot, supporting the conformational nature of this epitope. Positive immunoblot reactions with the SmD1 polypeptide are not inhabitable by the synthetic SmD1-aa83-119 peptide. Comparing anti-Sm reactivities detected by ELISAs with those in immunoblot, different patterns were observed, reflecting the heterogeneous autoimmune response to this antigen. In conclusion, the anti-SmD1-aa83-119 peptide ELISA substantially completes the panel of methods for autoantibody testing. As none of the assays presently available covers the whole spectrum of epitope specificities of anti-Sm Abs elicited in SLE, it does not replace traditional anti-Sm ELISAs.