Abstract

DEBESA PADILLA, Aymé  and  HERNANDEZ BETANCOURT, Oscar. Standardization of an immunoenzymatic assay to detect anti-double-stranded DNA antibodies in systemic lupus erythematosus. Rev Cubana Invest Bioméd [online]. 2012, vol.31, n.4, pp. 467-479. ISSN 0864-0300.

Introduction: anti-double-stranded DNA antibodies are a diagnostic serological marker for systemic lupus erythematosus (SLE). The solid-phase immunoenzymatic assay is a rapid, cost-effective technique for their detection. Objective: Standardize an ELISA detecting anti-double-stranded DNA for the diagnosis of lupus. Methods: the standardization process included the following steps: preparation of controls, sensitization of the solid phase, selection of buffers and assay conjugate, evaluation of reaction conditions and determination of the cut-off level. A study of unspecificities was also conducted. Five types of polystyrene plates were tested, and a comparison was made of E. coli (pUC19) plasmid DNA and human genomic DNA as coating antigens. An evaluation was conducted of the effect of poly (L-lysine) and irradiation of the plate with ultraviolet light upon antigen fixation. The assay cut-off value was determined by the limit value method. Results: antigen dissociation was observed when poly (L-lysine) was not used in the pretreatment of the plate and UV light irradiation did not foster DNA binding to the solid phase. No significant differences were found (p=0.710) between the two DNA coatings. The cut-off value (K=3) made it possible to classify 28 samples of patients with SLE as positive (63.6 %). Conclusions: the method standardized with the use of plasmid DNA enabled detection of anti-double-stranded DNA antibodies in patients with lupus.

Keywords : DNA; immunoenzymatic assays; ELISA; standardization.

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