Recognition of two distinct major antigens by bullous pemphigoid sera

Sera from 17 patients with bullous pemphigoid identified a range of polypeptides of relative molecular mass (Mr) 240,000, 230,000, 190,000, 180,000, 120,000 and 100,000 from extracts of SCaBER cells, cultured human keratinocytes or human epidermis, using an immunoblotting technique. The pattern of polypeptides was characteristic for the patient serum and individual sera identified similar polypeptides from all three substrates. All 17 sera recognized major polypeptides of either Mr 230,000 (11 sera) or Mr 180,000 (seven sera) under the denaturing conditions used for immunoblotting studies. Sera from 12 patients were also examined using an immunoprecipitation technique. Polypeptide(s) of Mr 230,000 were immunoprecipitated from extracts of SCaBER cells by 11 of these sera, despite immunoblotting patterns of Mr 180,000 (or less) for three of the 11 sera. None of the minor polypeptides recognized in immunoblotting studies were immunoprecipitated by these sera. Localization of antigens was determined by binding of sera to intact or permeabilized SCaBER cells in an ELISA. Sera which recognized the Mr 230,000 polypeptide under denaturing conditions also identified an intracellular epitope in SCaBER cells, while sera which identified the denatured Mr 180,000 polypeptide bound to a cell surface epitope. Two distinct major antigens are recognized by bullous pemphigoid sera. These both appear as molecules of Mr 230,000 under non-denaturing conditions, but only one of the molecules is dissociated to produce a Mr 180,000 polypeptide under denaturing conditions. Epitopes on these two major antigens are localized on either side of the cell membrane.