title: A highly sensitive and simple assay for the detection of circulating autoantibodies against full-length bullous pemphigoid antigen 180.
abstract: Bullous pemphigoid antigen 180 (BP180) is the target of autoantibodies in various subepidermal blistering diseases. The most common one is bullous pemphigoid (BP). The pathological importance of anti-BP180 antibodies has been demonstrated in a passive transfer mouse model. However, sensitive assays for routinely detecting circulating antibodies directed against both intra- and extracellular domains of BP180 are only available in specialized laboratories. In addition, most current assays use prokaryotic recombinant fragments of BP180 that lack conformation-dependent epitopes. A simple and very sensitive immunofluorescence (IF) assay based on eukaryotic cells is described here. Sf21 insect cells were transfected with full-length (FL) BP180. As revealed by FACS and confocal laser scanning microscopy the protein was expressed as type II transmembrane protein as in human keratinocytes. By testing serial dilutions of BP180-specific mouse monoclonal antibodies, the eukaryotic IF assay was demonstrated to be more sensitive compared to conventional assays including (1) indirect IF microscopy of human salt-split skin, (2) Western blotting (WB) of the keratinocyte-derived BP180 ectodomain, (3) WB of recombinant BP180 NC16A, and (4) WB of FL-BP180 extracted from Sf21 insect cells. When applied to sera from patients with BP (n = 65), pemphigoid gestationis (n = 16), and cicatricial pemphigoid (n = 7), the novel assay revealed that 58 (89%), 13 (81%), and 6 (84%), respectively, were positive. In contrast, all control sera (pemphigus, n = 20; epidermolysis bullosa acquisita, n = 5; anti-laminin 5 cicatricial pemphigoid, n = 5; systemic lupus erythematosus, n = 5; atopic dermatitis, n = 7; contact dermatitis, n = 3; normal human sera, n = 30) were negative indicating that the assay is highly specific. In addition, reactivity of the assay was conserved to a large extent when the cells had been stored at -20 degrees C for 3 months. Thus, this assay meets the demands of a simple and effective diagnostic tool for detecting circulating antibodies against FL-BP180 and may also be used in laboratories without access to molecular biological technology.
authors.affiliation: Department of Biotechnology, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Schmidt
authors.forename: Arno
authors.initials: A
authors.lastname: Kromminga
authors.forename: Saskia
authors.initials: S
authors.lastname: Mimietz
authors.forename: Ulrich
authors.initials: U
authors.lastname: Leinfelder
authors.forename: Cassian
authors.initials: C
authors.lastname: Sitaru
authors.forename: Eva-Bettina
authors.initials: EB
authors.lastname: Bröcker
authors.forename: Detlef
authors.initials: D
authors.lastname: Zillikens
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zimmermann
mesh_terms: D000368:Aged; D000818:Animals; D001323:Autoantibodies / Q000032:analysis* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D001327:Autoimmune Diseases / Q000175:diagnosis / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D002352:Carrier Proteins; D002478:Cells, Cultured; D003094:Collagen / Q000276:immunology*; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007313:Insecta; D008297:Male; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010390:Pemphigoid, Benign Mucous Membrane / Q000276:immunology; D010391:Pemphigoid, Bullous / Q000276:immunology; D011817:Rabbits; D012680:Sensitivity and Specificity; D000097805:Collagen Type XVII
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII; C486268:DST protein, human