Development and validation of serological immunoassays in laminin γ-1 pemphigoid and epidermolysis bullosa acquisita

Pemphigoid diseases encompass a heterogeneous group of subepidermal autoimmune bullous diseases of the skin and mucosa. These disorders are characterized by autoantibodies that target distinct structural proteins of the basement membrane zone which are crucial for the integrity of the skin. Targeting of these proteins leads to skin fragility, detachment of the epidermis and eventually formation of blisters and skin inflammation. The clinical presentation of the pemphigoids is heterogeneous, the subtypes are divided by clinical symptoms, target antigens and response to treatment. A crucial step for the proper classification is the serological detection of the autoantigens directed against distinct components of the dermal epidermal junction. Thus, it is crucial to develop reliable and reproducible diagnostic methods which help clinicians to differentiate between different entities and therefore to select the best treatment. This thesis is focused on Laminin γ-1 pemphigoid and epidermolysis bullosa acquisita, the study of their clinical appearance and the development and validation of serological assays to correctly diagnose these two entities. Both autoimmune blistering diseases are rare and often hard to diagnose with standard serological assays. In the first study presented in this thesis, I discuss the development of an immunoblot assay for the detection of autoantibodies against Laminin γ-1. Laminin γ-1 pemphigoid is a novel subepidermal blistering disease which has been first described in 1996. Since then, different immune serological methods for the detection of circulating autoantibodies have been described, however these are often not reproducible for most of the centers worldwide, since often advanced lab skills are required. In our study, we present a novel immunoblot assay based on three commercially available Laminin γ-1 recombinants, which can be reproduced in almost every laboratory with basic skills. In the second study, we thought to serologically identify for the best immunoassay for the detection of serum IgG autoantibodies against anti-human collagen VII in patients with epidermolysis bullosa acquisita. Epidermolysis bullosa acquisita is a severe blistering diseases characterized by circulating IgG autoantibodies targeting human collagen VII. In this study, four different diagnostic assays (immunoblot, indirect immunofluorescence with saline split human skin and 2 enzyme linked immunosorbent assays) have been tested and compared with regard to their sensitivity and specificity in a large cohort of epidermolysis bullosa acquisita sera (n=95). In summary, our study showed that enzyme linked immunosorbent assays based on recombinant human collagen VII NC1-NC2 or collagen VII-NC1 proteins provide the highest sensitivity and specificity for the detection of anticollagen VII IgG and should be preferred to immunoblot analysis or indirect immunofluorescence on saline split human skin in making the diagnosis of epidermolysis bullosa acquisita