Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid

Sera from 20 patients with bullous pemphigoid (BP) were analyzed for the subclass distribution of IgG autoantibodies by Western immunoblotting of normal human heat-separated epidermal extracts. Strips were sequentially probed with patient sera, monoclonal antibodies (Mab) anti-human IgG1-4, and a labeled anti-mouse Ig. The relative dilutions of each subclass-specific Mab were determined in dot immunobinding experiments in order to give uniform reactivity to the corresponding human IgG subclass. Circulating BP autoantibodies reacting with the major BP antigen of 220 kDa and/or a 165-kDa band showed a stricking predominance of IgG4. Overall, IgG4 was the major subclass reactive with the 220-kDa band, whereas, in addition to IgG4, IgG1 and IgG2 were frequently represented in the response to the 165-kDa band. Complement fixation of circulating BP antibodies was studied by complement indirect immunofluorescence using human serum as source of complement and FITC goat anti-human C3, C1q, and C4, on normal human skin substrate. When circulating BP antibodies contained IgG1 or IgG2 in addition to IgG4, they fixed C3 and, in some cases, C1q and C4, suggesting complement activation by either the classical or the alternate pathway.