Selective detection of autoimmune antibodies to single- and double-stranded DNA by enzyme immunoassay

Abstract

Presence in serum of anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) is one of the diagnostic criteria for systemic lupus erythematosus. Anti-single stranded DNA antibodies (anti-ssDNA) also occur, but their clinical significance is unclear. Use of enzyme immunoassay (EIA) kits that are specific for anti-dsDNA is desirable but problematic, since preparation of dsDNA of high integrity is difficult (ie, regions of ssDNA may persist). This study evaluated an EIA kit that uses low Mw plasmid/bacteriophage DNA as a nucleic acid source. Anti-dsDNA results were compared to results obtained by an anti-ssDNA EIA and an immunofluorescent assay (IFA) that uses Crithidia luciliae (specific for anti-dsDNA). Consecutive serum samples (n=139, 88% female) submitted to the clinical laboratory for anti-dsDNA analysis were evaluated. EIA precision was determined at three levels. Intra-assay precision [mean +/- SD (CV)] for anti-dsDNA: 36 +/- 3.5 IU/ml (9.8%); 98 +/- 4.4 (4.5%); and 245 +/- 5.8 (2.4%); and anti-ssDNA. 40 +/- 1.4 U/ml (3.5%); 190 +/- 4.8 (2.5%); and 283 +/- 10.3 (3.7%) (n=8). Inter-assay precision for anti-dsDNA: 36 +/- 6.3 IU/ml (17.5%); 90 +/- 5.9 (6.6%); and 207 +/- 20.9 (10.1%); and anti-ssDNA: 48 +/- 8.2 U/ml (17.0%); 193 +/- 12.7 (6.6%); and 263 +/- 21.5 (8.2%) (n=8). Linearity was assessed with (a) high dsDNA/high ssDNA and (b) low dsDNA/high ssDNA samples (no high dsDNA/low ssDNA samples were identified). Linearity (>200 IU/ml) was found for both sample types with a correlation coefficient (r) of 0.995-0.999. Anti-dsDNA immunoreactivity was not apparent with the low dsDNA/high ssDNA sample. More patients were positive for anti-ssDNA (54%), compared to anti-dsDNA (17%). IFA confirmation (Crithidia) indicated a relative sensitivity and specificity of 94.1% and 93.4% for the anti-dsDNA EIA. IFA positivity correlated with increased anti-dsDNA level: 20% (30-60 IU/ml); 70% (61-200 IU/ml); and 89% (>200 IU/ml). Of specimens that were anti-ssDNA positive and anti-dsDNA negative (n=51), only one was IFA positive. When IFA was compared to an anti-dsDNA EIA kit that used high Mw calf thymus DNA, lower relative specificity (88.2%) and sensitivity (72.6%) was obtained. Anti-ssDNA was found in many false positive specimens (87%).