Antibodies to centromere antigens measured by an automated enzyme immunoassay.

BACKGROUND: Anticentromere antibodies (ACA) are frequently observed in patients with Raynaud's phenomenon and in the CREST syndrome, a subclass of systemic sclerosis. Likewise, ACA are also found in other autoimmune and non-autoimmune diseases. The objective of the present study was to evaluate the clinical utility of the measurement of antibodies to the best characterized centromere antigen (CENP-B) protein by an enzyme-linked immunosorbent assay (ELISA) that uses human recombinant CENP-B antigen and compare it with indirect immunofluorescence assay (IFA) on HEp-2 cells. METHODS: We have analyzed 128 sera samples from patients with the following diseases: systemic lupus erythematosus (SLE, n = 53), mixed connective tissue disease (n = 1), primary Sjögren syndrome (n = 10), primary Raynaud's phenomenon (n = 10), primary systemic sclerosis (n = 7), polymyositis/dermatomyositis (n = 3), rheumatoid arthritis (n = 9), cutaneous lupus (n = 5), primary biliary cirrhosis (n = 9), chronic autoimmune hepatitis (n = 5) and ANA-positive non-autoimmune diseases (n = 16). RESULTS: The ELISA evaluated shows a good concordance with IFA, with the advantage of being an automatable quantitative technique. CONCLUSIONS: Measurement of anticentromere antibodies by this ELISA using human recombinant antigen is a useful alternative for the autoimmune laboratory checking for diseases associated with anticentromere antibodies.

Antibodies to fibrillarin, PM-Scl and RNA polymerase III detected by ELISA assays in patients with systemic sclerosis.

BACKGROUND: Anti-fibrillarin (AFA), anti-RNA polymerase (anti-RNAP), and anti-PM-Scl autoantibodies are useful markers for the diagnosis of systemic sclerosis (SSc) in patients who are anti-centromere- (ACA) or anti-topoisomerase I (anti-topo I)-negative, but, until recently, the only specific method for their identification was the radio-immunoprecipitation assay. The aim of this study was to evaluate the clinical accuracy of the new enzyme-linked immunosorbent assays (ELISA) developed by Phadia for their detection. METHODS: Sera of 50 ACA and anti-topo I-negative SSc patients, and, as control group, sera of 122 patients (42 with SSc, ACA or anti-topo I-positive, 40 with systemic lupus erythematosus and 40 with rheumatoid arthritis) were studied. RESULT: Using the cutoff proposed by the manufacturer (10 AU/mL), sensitivity and specificity were: for AFA, 22% and 92.6%; for anti-RNAP, 16% and 97.5%; and for anti-PM-Scl, 8% and 98.8%, respectively. Using a cutoff corresponding to 98.8% specificity for all three antibodies, sensitivity was 10%, 14% and 8%, respectively. The combined use of these three antibody assays enabled identification of 32% of ACA- and anti-topo I-negative SSc patients. CONCLUSIONS: These new ELISA methods for AFA, anti-RNAP III and anti-PM-Scl detection have good diagnostic specificity, and may help identify a subset of SSc patients ACA and anti-topo I-negative.

Analytical and diagnostic accuracy of the EliA automated enzyme fluoroimmunoassay for antineutrophil cytoplasmic autoantibody detection.

Anti-proteinase 3 antineutrophil cytoplasmic antibodies (PR3-ANCA) and anti-myeloperoxidase antibodies (MPO-ANCA) are considered important serological markers for several forms of idiopathic systemic vasculitis. The aim of the study was to verify the analytical and clinical performance of a new automated enzyme fluoroimmunoassay, the EliA system, for PR3-ANCA and MPO-ANCA detection. For this purpose the sera of 52 consecutive well-defined patients with a clinical diagnosis of Wegener's granulomatosis (WG) (n=29) or microscopic polyangiitis (MPA) (n=23), and 70 controls suffering from connective tissue disease (25 systemic lupus erythematosus, 25 Sjögren's syndrome and 20 rheumatoid arthritis) were tested for PR3-ANCA and MPO-ANCA with the EliA assay (Pharmacia Diagnostics, Freiburg, Germany). For comparison purposes, the same sera were also tested by indirect immunofluorescence, another direct immunometric assay (Varelisa, Pharmacia Diagnostics) and a capture PR3-ANCA (Wieslab AB, Lund, Sweden) method. Both the EliA PR3-ANCA and MPO-ANCA assays showed between- and within-assay precision of <10%. The dilution test gave straight lines (r2=0.998) for both antibody assays. The recovery ranged from 97.9% to 102.7% for PR3-ANCA and from 84.9% to 91.4% for MPO-ANCA. There was a high positive correlation between the EliA and Varelisa methods for quantitative detection of MPO-ANCA levels (r2=0.949) and a lower correlation for PR3-ANCA (r2=0.771). Conversely, poor correlation was observed between EliA PR3-ANCA and capture PR3-ANCA (r2=0.537). The overall sensitivity and specificity of EliA PR3-ANCA and MPO-ANCA for the vasculitides considered in this study were 82.7% and 97.2%, respectively, with a positive predictive value of 96.6% and a negative predictive value of 84.9%. Comparison of the results obtained with the indirect immunofluorescence, Varelisa and capture PR3-ANCA methods showed that the indirect immunofluorescence assay is the most sensitive method for the diagnosis of vasculitis (88.5%), but the least specific (94.3%); the EliA method is slightly more specific (97.2%) than the Varelisa method (95.7%), and also slightly more sensitive (82.7% vs. 80.8%). Capture PR3-ANCA proved to be the most sensitive method for detection of anti-proteinase 3 antibodies in WG (89.7% vs. 86.2% EliA and 79.3% Varelisa). In conclusion, the EliA MPO-ANCA and PR3-ANCA methods provide good diagnostic accuracy and excellent analytical accuracy, which, in association with the practicality of the automated EliA system, make this method a useful tool for the diagnosis of ANCA-associated vasculitides.

Clinical evaluation of a new automated anti-dsDNA fluorescent immunoassay.

The measurement of anti-double-stranded DNA (anti-dsDNA) antibodies is a useful tool for the diagnosis and the follow-up of systemic lupus erythematosus (SLE). Anti-dsDNA antibodies are involved in the pathogenesis of lupus nephritis and they are, specially the high-avidity antibodies, the most specific antibodies associated with SLE nephritis and active SLE. The aim of the present study was to assess the clinical utility of an enzyme-linked immunosorbent assay (EUSA) that utilizes a circular double-stranded plasmid DNA as a nucleic acid source, adapted to an automated fluorescence immunoassay (EliA dsDNA, Pharmacia, Freiburg, Germany). Also, we compared this method with other immunoassays used in clinical laboratories. We have measured anti-dsDNA antibodies in the serum of 179 patients with a positive result for antinuclear antibodies (ANA). Seventy six sera were from SLE patients (14 men and 62 women), and the other 103 sera (from 20 men and 83 women) constituted the control group. This latter group includes nine Sjogren's syndrome patients, six patients with rheumatoid arthritis and 88 with various other diseases, including connective tissue diseases (n=34), hepatopathies (n= 17; 11 primary biliary cirrhosis and 6 autoimmune hepatitis), and 37 patients with nonautoimmune diseases (viral hepatitis, renal disease, diabetes, exanthema and hypertension). Methods used were "EliA dsDNA" (Pharmacia, Germany), "Varelisa dsDNA" (Pharmacia, Germany), Farr (Amersham, UK) and Chritidia luciliae immunofluorescence test (Vitro-Immun, Germany). We assessed sensitivity, specificity, positive predictive value and negative predictive value in the clinical study, and kappa index and scatter plots in the comparative study. The results show a low concordance between methods (kappa < 0.6). The evaluated EliA method shows a very good specificity for SLE (93.2%) and a good sensitivity for active SLE (70.8%).