Fig. 3

Detection of autoantibodies by ELISA with extracellular matrix of cultured keratinocytes. The extracellular matrix was obtained by treatment of cultured HaCaT keratinocytes with 20 mM NH4OH as detailed in Methods. a SDS-PAGE analysis of the extracellular matrix extract shows the migration of laminin 332 chains (arrows) at about 165 kDa (α3 chain), 140 kDa (β3 chain) as well as 155 kDa and 105 kDa (unprocessed and processed forms of the γ2 chain). b Immunoreactivity of mucous membrane pemphigoid autoantibodies with the laminin 332 from the extracellular matrix extract. Extracellular matrix extract was electrophoretically separated by 8% SDS-PAGE, transferred to nitrocellulose and immunoblotted with rabbit anti-human laminin 332 polyclonal antibody (lane 1), anti-laminin 332 mucous membrane pemphigoid patient’s sera (lanes 2–4), normal human sera (lane 5), and normal rabbit sera (lane 6). c Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. Test performed with sera from patients with confirmed anti-laminin 332 mucous membrane pemphigoid (n = 36) and controls (n = 116). d ELISA reactivity of human sera with extracellular matrix. Scatter plots represent optical density measurements of serum reactivity from patients with mucous membrane pemphigoid (MMP; n = 36) with confirmed laminin 332-specific autoantibodies, MMP patients with possible laminin 332-specific autoantibodies (n = 30), from healthy donors (NHS; n = 116), as well as from patients with bullous pemphigoid (BP; n = 89), pemphigus vulgaris (PV; n = 49), epidermolysis bullosa acquisita (EBA; n = 19), and dermatitis herpetiformis (DH; n = 41). The cut-off of the assay is represented by a dotted line (cut-off = 0.367, sensitivity = 83.33%, specificity = 84.48%)