Development of a system for detection of circulating antibodies against hemidesmosomal proteins in patients with bullous pemphigoid

Specific antibodies directed against special hemidesmosomal proteins are in volved in the pathogenesis of bullous pemphigoid (BP), and detection of the se antibodies is crucial for a correct diagnosis. As the BP autoantigen pri mary structures are known, the question was addressed as to whether it is p ossible to demonstrate circulating antibodies against BP autoantigens (BPAG 1 and BPAG2) by means of an ELISA system, using antigenic epitopes. With th e help of the programs Peptidestructure and Plotstructure, antigenic epitop es of BP antigens were predicted, chemically synthesized and screened using serum from 43 proven BP patients. The coding sequences of the best antigen ic epitopes were then chemically synthesized and inserted as monomer and ho mo- or hetero-oligomer forms into fusion-expression plasmids (PGEX-4T, Phar macia) in-frame to the C-terminus of glutathione-S-transferase. Fusion prod ucts were expressed and purified from Escherichia coli cells by affinity ch romatography. The recombinant proteins were used for the detection of antib odies in the serum of 43 BP patients and of 60 controls (including 30 healt hy persons, 22 patients with pemphigus vulgaris and 8 patients with other b ullous dermatoses). Use of the homo- and hetero-oligomers of the recombinan t fusion peptides increased the sensitivity of the disease-specific antibod y detection. When a mixture of the best recombinant fusion proteins was use d, the sensitivity of the ELISA assays in the case of the BP patients' seru m was 0.90. This system could form the basis of a rapid and simple system f or the diagnosis of BP.