BP180 (type XVII collagen, Col17) is a main target antigen in the two subepidermal blistering diseases bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP). The NC16A domain is targeted in 80-90% of BP and 50% of MMP sera and is applied in highly sensitive and specific widely available ELISA systems. Additionally, epitopes on the BP180 ectodomain outside NC16A have been reported. So far, for a considerable number of BP and MMP patients, no standardized assay is available for the detection of non-NC16A anti-BP180-specific autoantibodies. Here, four overlapping His-tagged proteins covering the BP180 ectodomain (F1, amino acids (aa) 563-807, F2, aa793-1039, F3, aa1024-1270, F4, aa1255-1497) outside the NC16A domain were cloned, expressed, purified and analyzed for their reactivity with patient sera by immunoblotting (IB). Only NC16A-unreactive BP (n=50) and MMP (n=30) sera with reactivity to the cell-derived soluble ectodomain of BP180 (LAD-1) were included. As controls, sera of healthy blood donors (n= 50) were used. 98% of BP and 77% of MMP sera exhibited autoantibodies against F3 (specificity 98%), followed by reactivity with F4 (70% of BP and 63% of MMP sera, 89% specificity), F2 (42% of BP and 53% of MMP sera, 89% specificity) and F1 (20% of BP and 13% of MMP, 91% specificity). Additionally, autoantibody reactivity with recombinant full-length (FL) BP180 and the BP180 ectodomain (BP180ec, including NC16A) was analyzed. 100% of BP and 73% of MMP sera reacted with FL BP180, whereas 98% of BP and 87% of MMP sera showed reactivity with BP180ec (94% specificity for both tests). In this study, a 246 aa stretch in the C-terminal part of BP180 was identified, that may be applied for the development of a widely used test system for the detection of non-NC16A-reactive anti-BP180 antibodies. This assay will close a considerable gap in the serological diagnosis of BP and MMP.

Sera of patients with bullous pemphigoid and mucous membrane pemphigoid react with a C-terminal 246 amino acid stretch of BP180