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. 2004 Apr;63(4):386–394. doi: 10.1136/ard.2003.016303

Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity

K Haugbro 1, J Nossent 1, T Winkler 1, Y Figenschau 1, O Rekvig 1
PMCID: PMC1754943  PMID: 15020332

Abstract

Background: The presence of "anti-DNA antibodies in abnormal titres" is a well established criterion for SLE classification, but there is no agreement on the performance of this test.

Objective: To study the correlation between clinical findings and five different solid and solution phase anti-DNA antibody assays.

Methods: 158 consecutively collected ANA positive sera were studied in a double blind fashion. Anti-DNA antibodies were determined by different solid phase assays (ssDNA-, dsDNA- specific ELISA, EliA anti-dsDNA assay, Crithidia luciliae assay), and by an experimental solution phase anti-DNA assay using biotinylated pUC18 plasmid, human, calf thymus, and E coli DNA. Antibody affinity was determined by surface plasmon resonance. Clinical data were obtained independently of the laboratory analyses and later related to the anti-dsDNA findings.

Results: Anti-dsDNA antibodies were most frequently detected by ELISA, but were not specific for SLE as they were present in up to 30% of other disease groups. Those detected by the Crithidia luciliae assay were predictive for SLE, while antibodies binding in solution phase ELISA using the pUC18 correlated strongly with the Crithidia luciliae assay. Surface plasmon resonance analysis showed that antibody binding to pUC18 was not due to higher relative affinity for dsDNA in general, but apparently to specificity for that plasmid DNA. Serum samples from three patients with lupus nephritis were positive in both pUC18 solution phase and Crithidia luciliae assays.

Conclusions: Assay principle selection is decisive for the detection of clinically significant anti-DNA antibodies. Revision of the anti-DNA antibody criterion in the SLE classification may be needed.

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Figure 1 .

Figure 1

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Consecutively collected ANA positive sera were examined for anti-DNA antibodies by different solid phase assays. Correlations are shown for anti-ssDNA and anti-dsDNA as detected by solid phase ELISA (rs = 0.4, p = 0.002) (A); anti-dsDNA antibodies detected in dsDNA ELISA versus CLIFT (rs = 0.11, p = 0.4) (B); dsDNA ELISA versus EliA dsDNA assay (rs = 0.55, p = 0.001) (C); and CLIFT versus EliA dsDNA assays (rs = 0.58, p<0.0001) (D). Inserted in each figure (A–D) are 2x2 tables showing the number of patients. The cut off values are 55 (U), 20 (U), and 10 (titre), for ELISA ssDNA/dsDNA, EliA dsDNA and CLIFT, respectively.

Figure 2 .

Figure 2

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Groups of sera, selected as ELISA anti-dsDNA antibody positive, CLIFT negative (n = 8, A, B); anti-dsDNA antibodies positive in ELISA as well as in CLIFT (n = 12, C-E); or ANA positive, anti-ssDNA/dsDNA antibody negative sera (n = 5, F), were analysed in solution phase, biotinylated DNA ELISA (SPADE) using human, CT, E coli ssDNA/dsDNA or circular pUC18 dsDNA as antigens. Mean OD 490 (SD) for each group of sera at each serum dilution is presented for antibody binding to human dsDNA and ssDNA, and pUC18 circular and linear dsDNA (A, C, F). Correlation between CLIFT and relative pUC18 titres in all 25 sera included in these analyses is presented (E). Mean titres (SD) of anti-DNA antibodies from CLIFT negative (B) and CLIFT positive (D) sera against circular pUC18 dsDNA, human, calf thymus or E coli ssDNA/dsDNA demonstrate that in CLIFT positive sera, but not negative ones, antibodies are detected that recognise structures unique to pUC18 dsDNA (B, D).

Figure 3 .

Figure 3

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Surface plasmon resonance sensograms obtained at different concentrations of IgG anti-DNA antibodies interacting with immobilised 32 bp oligonucleotide or circular pUC18 dsDNA. IgG anti-DNA antibodies from CLIFT positive serum 160 binds both DNA ligands, while those from CLIFT negative serum 135 bind the oligonucleotide, but not pUC18. See table 1 for extended data, and "Materials and methods" for experimental details.

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