Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid

Abstract

Anti-p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200-kDa protein (p200) of the dermal-epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C-terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti-p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L(-1) salt-split skin by indirect immunofluorescence microscopy and labelling of a 200-kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories. To develop a simple, sensitive and specific diagnostic tool for anti-p200 pemphigoid. Sera from patients with anti-p200 pemphigoid (n = 35), bullous pemphigoid (BP, n = 101), epidermolysis bullosa acquisita (EBA, n = 10), antilaminin 332 mucous membrane pemphigoid (MMP, n = 14), pemphigus vulgaris (PV, n = 51) and healthy volunteers (HV, n = 131) were tested by a novel enzyme-linked immunosorbent assay (ELISA) that employed a recombinant monomeric C-terminal fragment of human laminin γ1 (hLAMC1-cterm) expressed in Escherichia coli. Serum reactivity with hLAMC1-cterm was detected in sera from 24 of 35 (69%) patients with anti-p200 pemphigoid, two of 101 (2%) with BP, 0 of 10 with EBA, two of 14 (14%) with anti-laminin 332 MMP, 0 of 51 with PV, and 0 of 131 HV. This novel ELISA will facilitate the diagnosis of anti-p200 pemphigoid.