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 involved in the pathogenesis of bullous pemphigoid (BP), and detection of these antibodies is crucial for a correct diagnosis. As the BP autoantigen primary structures are known, the question was addressed as to whether it is possible to demonstrate circulating antibodies against BP autoantigens (BPAG1 and BPAG2) by means of an ELISA system, using antigenic epitopes. With the help of the programs Peptidestructure and Plotstructure, antigenic epitopes of BP antigens were predicted, chemically synthesized and screened using serum from 43 proven BP patients. The coding sequences of the best antigenic epitopes were then chemically synthesized and inserted as monomer and homo- or hetero-oligomer forms into fusion-expression plasmids (PGEX-4T, Pharmacia) in-frame to the C-terminus of glutathione-S-transferase. Fusion products were expressed and purified from Escherichia coli cells by affinity chromatography. The recombinant proteins were used for the detection of antibodies in the serum of 43 BP patients and of 60 controls (including 30 healthy persons, 22 patients with pemphigus vulgaris and 8 patients with other bullous dermatoses). Use of the homo- and hetero-oligomers of the recombinant fusion peptides increased the sensitivity of the disease-specific antibody detection. When a mixture of the best recombinant fusion proteins was used, the sensitivity of the ELISA assays in the case of the BP patients' serum was 0.90. This system could form the basis of a rapid and simple system for the diagnosis of BP.
Husz
S
S
Department of Dermatology, Albert Szent-Györgyi Medical University, Szeged, Hungary. hs@derma.szote.u-szeged.hu
Kiss
M
M
Molnár
K
K
Marczinovits
I
I
Molnár
J
J
Tóth
G K
GK
Dobozy
A
A
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins; D003001:Cloning, Molecular; D003094:Collagen / Q000276:immunology*; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000097:blood / Q000276:immunology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology; D000097805:Collagen Type XVII
D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D000939:Epitopes; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII; C486268:DST protein, human