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Characterization of the humoral immune response in the pemphigoids and development of novel diagnostic BP180, BP230 and laminin 5 ELISA assays

The pemphigoids (comprising bullous pemphigoid, gestational pemphigoid and mucous membrane pemphigoid) are a group of autoimmune bullous disorders of the skin and mucous membranes characterised by defective epithelial adhesion and by circulating and tissue-bound autoantibodies against components of the cutaneous basement membrane zone. Pemphigoid patient autoantibodies preferentially target two components of the epithelial adhesion structures called hemidesmosomes: the BP180 antigen (or collagen XVII), a type II collagenous transmembrane protein, and the cytoplasmic BP230 antigen. In particular, the majority of patients present autoantibodies against the NC16A subdomain of BP180 ectodomain. The third major antigenic target in pemphigoid patients is the epithelial adhesion ligand laminin 5. In this project, we have characterized the humoral immune response in the pemphigoids and developed novel diagnostic ELISAs.
Specifically, we have obtained:
%1) nine ELISAs based on different regions of BP180 (five different regions of the ectodomain and also the entire ectodomain) and BP230 (three regions spanning almost the entire antigen), produced as baculovirus-derived recombinant proteins, which were validated testing large cohorts of bullous pemphigoid patients sera. Overall these ELISAs displayed a high sensitivity and specificity. However and in keeping with current concepts on disease pathogenesis, the global diagnostic properties of the BP180–ELISA outperformed those of the BP230-ELISA. These ELISAs also allowed to correlate antigen reactivity and clinical phenotypes and to delineate markers for disease activity and course;

2) six ELISAs based on BP180 regions produced as prokaryotic recombinant proteins that were used to detect intramolecular epitope spreading events during the disease course and to improve the diagnostic performance of the commercially available ELISA based on NC16A alone. These ELISAs were based on epitopes selected by screening a BP180 random epitope lambda library with bullous pemphigoid patient’s sera.

3) an ELISA based on native laminin 5 purified from the conditioned medium of a squamous carcinoma cell line. When used to screen a large cohort of pemphigoid patients sera, our ELISA detected anti-laminin 5 reactivity in the majority of mucous membrane pemphigoid sera and also in a part of bullous pemphigoid sera tested. Moreover, the titers of laminin 5-specific IgG correlated with clinical severity of mucous membrane pemphigoid. Collectively our findings show that these novel ELISAs represent a highly sensitive and specific assay for the rapid diagnosis of the pemphigoids and may provide predictive parameters for the management of pemphigoid patients.

Informations connexes

Contact

Michael HERTL, (Professor and Chairman)
Tél.: +49-642-12866280
Fax: +49-642-12862902
E-mail