Epidermolysis bullosa acquisita (EBA) is a severe blistering skin disease in which the epidermis separates from the dermis through the basement membrane zone (BMZ). Blister formation within the BMZ begins beneath the lamina densa where autoantibodies are deposited. Using autoantibodies from EBA patients' sera, we identified the EBA antigen as a large matrix molecule of 290,000 daltons that is distinct from bullous pemphigoid antigen, laminin, elastin, fibronectin and collagens I, IV and V. Our current goal is to purify and further characterize the EBA antigen using both conventional protein chemistry and molecular biology. 0.4 mm keratotomed slices of human skin will be de-epidermized in 1M salt at 4 degrees C for 96 hours, the epidermis discarded and the dermal BMZ extracted in 6M GuHC1, 0.05 M Tris-HC1, pH 9.2, 0.002 EDTA, with reducing agents. Proteins below 100 kd will be removed by dialysis and ultrafiltration. Collagens will be separated from EBA antigen by chromatography on a phenyl boronate affinity column. EBA antigen will be further purified by DEAE ion exchange and molecular sieve chromatography. If further purification is needed, hydroxyapatite chromatography, affinity chromatography on a fibronectin column and an anti-EBA antigen monoclonal antibody column will be attempted. An ELISA assay will be developed for rapid antigen and antibody detection. Purified EBA antigen will be characterized by isoelectric focusing, protease mapping, glyconse mapping, hexosamine determination, amino acid analysis, molecular sieve chromatography for native size, and rotart shadowing for molecular shape. In parallel, EBA antigen purification and characterization will be attempted by recombinant DNA technology: antigen blotted to activated glass filters will be placed in a gas-phase microsequanator. With an amino acid sequence, a corresponding balanced oligonucleotide probe will be synthesized and labeled and a human skin fibroblast cDNA library will be screened. Probe positive clones will be cultured and DNA purified. Positive identification of the clones will include transcribing RNA from the positive cDNA clones or isolating it by positive selection. This newly synthesized mRNA will be used in a rabbit reticulocyte cell-free translation system to make product (EBA antigen) which will be verified by SDSPAGE, blotting, immunoprecipitation and amino acid sequencing.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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General Medicine A Subcommittee 2 (GMA)
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University of North Carolina Chapel Hill
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