Epidermolyis bullosa acquisita (EBA) is a severe blistering disease of the skin characterized by separation of the epidermis from the dermis through the basement membrane zone (BMZ). Within the BMZ, immunoreactants are deposited immediately below the lamina densa, the site where blister formation begins. Using anti-BMZ auto-antibodies from EBA patients' sera, we identified the EBA antigen in human skin BMZ extracts by gel electrophoresis and Western immune blots as a large matrix macromolecule of 290,000 daltons that is distinct from bullous pemphigoid antigen, laminin, fibronectin, and collagen types I, IV and V. We now wish to purify and characterize the EBA antigen. O.4 mm keratotomed slices of skin will be de-epidermized in 1 M salt at 4 C for 96 hours, the epidermis discarded and the BMZ of the remaining dermis extracted in 8 M urea, 0.05 M Tris, pH 6.8 with reducing agents. Proteins below 100 kd will be removed by dialysis and ultrafiltration. Proteins above 100 kd will be separated by ion exchange chromatography, molecular sieve chromatography and hydroxylapatite chromatography. If further purification is needed, affinity chromatography with known matrix molecule affinity columns, anti-EBA monoclonal antibodies, commercially available affinity resins, and textile industrial dyes will be attempted. Isoelectric focusing will be done to obtain the isoelectric point of EBA antigen and as a preparative step. In addition to detecting EBA antigen by Coomasie blue stained gels and Western immune blots, and ELISA assay will be developed for rapid detections. Purified EBA antigen will be characterized by staphylococcal aureas V8 protease mapping, molecular sieve chromatography for a native molecular size, rotary shadowing for molecular shape, and biosynthetic labeling studies of cultures to determine its chemical classification. Biological properties including its cell binding ability (attachment assay), interactions with known matrix molecules (antigen-to-antigen ELISA), protease susceptibilities, and ability to provide a BMZ permiability barrier will be accessed.
Showing the most recent 10 out of 82 publications
