A new oligonucleotide-based ELISA for the detection of anti-double-stranded DNA antibodies

Abstract

The detection and measurement of antibodies to double-stranded (ds) DNA is important in the diagnosis and monitoring of patients with systemic lupus erythematosus (SLE). Several methods are available for their determination but none of them is completely satisfactory. Usually, purified dsDNA from different sources is used as antigen in the solid (enzyme-linked immunosorbent assay, ELISA) or liquid phase (Farr assay). Alternatively, anti-dsDNA antibodies can be demonstrated by immunofluorescence, using the haemoflagellate Crithidia luciliae (CLIFT).

We have developed a new oligonucleotide-based anti-dsDNA ELISA in which dsDNA, constituted of a 5′photobiotinylated nucleic acid probe and its highly specific complementary oligonucleotide, is formed onto the solid-phase. To do that, a 40 bp probe is coated on the microplate wells via streptavidin–biotin bond and the in vitro (in solid-phase) developed hybrid between probe and its complementary helix used as capturing antigen. The assay has proved to be specific and the several experiments performed to ascertain the absence of single-stranded DNA (ssDNA) contamination and/or non specific binding to plastic, streptavidin, probe (without complementary chain) have all excluded any significant interference.

To evaluate the clinical performance of this new assay, 114 serum samples from 68 SLE patients and 85 samples from 85 disease controls, in addition to 30 blood donors, were studied. The results were compared with commercial ELISAs, Farr assay and indirect immunofluorescence.

The sensitivity and specificity were 66.2 and 96.4%, respectively, comparable and even better than those of the commercially available anti-dsDNA kits. Anti-dsDNA antibody levels, measured with the oligonucleotide–ELISA, correlated with SLE clinical activity determined using the European Consensus Lupus Activity Measurement (ECLAM) (r = 0.59, p < 0.0001).

In conclusion, this assay has proved to be reproducible, and the results correlate well with other standard anti-dsDNA assays. The features of this new assay make it promising for clinical use.

• Anti-dsDNA antibodies
• ELISA
• oligonucleotides
• systemic lupus erythematosus (SLE)

Acknowledgements

We wish to thank Dr B. Bollini and Dr C. Corace for their technical assistance, and Fondazione “D'Amico” per la Ricerca sulle Malattie Renali for support.