Enzyme-linked immunosorbent assay for detection of peptide-specific human antidesmoplakin autoantibodies

Abstract

Autoantibodies directed against desmoplakin (Dp) I and II have recently been characterized in a subset of patients with severe erythema multiforme (EM), a recurrent inflammatory skin disease with a broad spectrum of clinical manifestations. These autoantibodies recognize a peptide epitope localized within the extreme end of the carboxy terminal domain of Dp responsible for the assembly of keratin filaments to the desmosomal plaque. Using dot blot analysis with overlapping synthetic peptides, the binding epitope YSYSYS has been identified. To establish an enzyme-linked immunosorbent assay (ELISA) for detection of peptide-specific anti-Dp autoantibodies in sera of patients with EM. A synthetic peptide containing the respective amino acid sequence was used as matrix for ELISA plates. Serum samples from patients with known EM and peptide-specific anti-Dp autoantibodies verified by immunoblotting, immunoprecipitation and epitope mapping were used. Establishing an index value of 42.0, 25 of 25 serum samples from five patients with peptide-specific anti-Dp autoantibodies were positive in the ELISA. From control sera, none of 31 bullous disease sera and only one (1.2%) of 83 normal human sera were positive. These data show that the ELISA presented in this study represents a sensitive and highly specific tool for the detection of peptide-specific anti-Dp autoantibodies in patients with EM.