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2012, Number 4

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Rev Cubana Invest Bioméd 2012; 31 (4)

Standardization of an immunoenzymatic assay to detect anti-double-stranded DNA antibodies in systemic lupus erythematosus

Debesa PA, Hernández BO

Language: Spanish
References: 22
Page:
PDF size: 262.52 Kb.


ABSTRACT

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.


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