FIG. 1.

Mapping of the bullous pemphigoid (BP) IgE MAb reactive sites by immunoblot. (A) Supernatants of our 395 hybridoma, Clones A5 and D2, were screened for IgE reactivity to either affinity-purified NC16A protein or lysates of bacteria expressing one of five subregions: NC16A1, NC16A2, NC16A2.5, NC16A3, or NC16A1–3. Proteins or lysates were electrophoresed and transferred to a nitrocellulose membrane. Membranes were probed with hybridoma supernatants (diluted 1:2) as the primary antibody. Anti-human IgE was used to detect specific reactivity to purified NC16A protein (left panel) and the NC16A segment 2 subdomain (right panel). 395 clone A5 is shown. Similar results were obtained with 395 clone D2. (B) To ensure specificity, 395 clone A5 and 395 clone D2 were used as primary antibodies on protein blots containing equimolar amounts of NC16A-GST, GST, and NC16A segment 2, followed by secondary detection with either anti-mouse IgE or IgG. HD18 (control) is mouse IgG specific for NC16A.