TY - JOUR
T1 - A highly sensitive and simple assay for the detection of circulating autoantibodies against full-length bullous pemphigoid antigen 180
AU - Schmidt, Enno
AU - Kromminga, Arno
AU - Mimietz, Saskia
AU - Leinfelder, Ulrich
AU - Sitaru, Cassian
AU - Bröcker, Eva Bettina
AU - Zillikens, Detlef
AU - Zimmermann, Ulrich
N1 - Funding Information:
This work was supported by grants from the Bundes-ministerium für Bildung und Forschung (BMBF-16SV1329/0 to U.Z.) and the Interdisciplinary Center for Clinical Research at the University of Würzburg, Germany (IZKF-01KS9603 to E.S.). We thank Dr Katshushi Owaribe, Nagoya, Japan for providing us with monoclonal antibodies 233 and 1A8c to BP180 and 1E5 to BP230. We also thank Dr George J. Giudice, Milwaukee, USA that kindly provided us with rabbit antibody 136 to GST-BP180 4575.
PY - 2002/6
Y1 - 2002/6
N2 - 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°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.
AB - 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°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.
UR - http://www.scopus.com/inward/record.url?scp=0036592061&partnerID=8YFLogxK
U2 - 10.1006/jaut.2002.0589
DO - 10.1006/jaut.2002.0589
M3 - Journal articles
C2 - 12144811
AN - SCOPUS:0036592061
VL - 18
SP - 299
EP - 309
JO - Journal of Autoimmunity
JF - Journal of Autoimmunity
SN - 0896-8411
IS - 4
ER -
