Abstract
BackgroundAutoimmunity to collagen VII is typically associated with the skin blistering disease epidermolysis bullosa acquisita (EBA), but also occurs occasionally in patients with systemic lupus erythematosus or inflammatory bowel disease. The aim of our present study was to develop an accurate immunoassay for assessing the presence of autoantibodies against collagen VII in large cohorts of patients and healthy donors.MethodsBased on in silico antigenic analysis and previous wetlab epitope mapping data, we designed a chimeric collagen VII construct containing all collagen VII epitopes with higher antigenicity. ELISA was performed with sera from patients with EBA (n = 50), Crohn's disease (CD, n = 50), ulcerative colitis (UC, n = 50), bullous pemphigoid (BP, n = 76), and pemphigus vulgaris (PV, n = 42) and healthy donors (n = 245).ResultsBy ELISA, the receiver operating characteristics analysis yielded an area under the curve of 0.98 (95% CI: 0.9638-1.005), allowing to set the cut-off at 0.32 OD at a calculated specificity of 98% and a sensitivity of 94%. Running the optimized test showed that serum IgG autoantibodies from 47 EBA (94%; 95% CI: 87.41%-100%), 2 CD (4%; 95% CI: 0%-9.43%), 8 UC (16%; 95% CI: 5.8%-26%), 2 BP (2.63%; 95% CI: 0%-6.23%), and 4 PV (9.52%; 95% CI: 0%-18.4%) patients as well as from 4 (1.63%; 95% CI: 0%-3.21%) healthy donors reacted with the chimeric protein. Further analysis revealed that in 34%, 37%, 16% and 100% of sera autoantibodies of IgG1, IgG2, IgG3, and IgG4 isotype, respectively, recognized the recombinant autoantigen.ConclusionsUsing a chimeric protein, we developed a new sensitive and specific ELISA to detect collagen specific antibodies. Our results show a low prevalence of collagen VII-specific autoantibodies in inflammatory bowel disease, pemphigus and bullous pemphigoid. Furthermore, we show that the autoimmune response against collagen VII is dominated by IgG4 autoantibodies. The new immunoassay should prove a useful tool for clinical and translational research and should improve the routine diagnosis and disease monitoring in diseases associated with collagen VII-specific autoimmunity.
Highlights
Autoimmunity to collagen VII is typically associated with the skin blistering disease epidermolysis bullosa acquisita (EBA), and occurs occasionally in patients with systemic lupus erythematosus or inflammatory bowel disease
pemphigus vulgaris (PV) was diagnosed in patients presenting (a) intraepidermal skin blisters and mucosal or mucocutaneous involvement, (b) intercellular IgG deposits within the epidermis detected by direct immunofluorescence (DIF) microscopy, (c) Serum IgG autoantibodies binding to the epithelium of monkey esophagus with an intercellular pattern by IF microscopy, and (d) IgG autoantibodies against desmoglein 3 by ELISA
In silico prediction of B cell epitope Wet lab epitope mapping studies using different recombinant fragments of collagen VII showed that the epitopes targeted by autoantibodies are localized within the NC1, NC2 and, possibly, the hinge region of the antigen
Summary
Autoimmunity to collagen VII is typically associated with the skin blistering disease epidermolysis bullosa acquisita (EBA), and occurs occasionally in patients with systemic lupus erythematosus or inflammatory bowel disease. An immune response against collagen VII is typically associated with epidermolysis bullosa acquisita (EBA) and bullous systemic lupus erythematosus, but may occur in other conditions, including inflammatory bowel disease (IBD) and dystrophic epidermolysis bullosa [1,2]. Large part of the NC2 domain is proteolytically removed by bone morphogenetic protein 1 (BMP-1), an enzyme with procollagen C-proteinase activity In spite of this proteolytic cleavage a small peptide of NC2 domain consisting of 41 aminoacids still reside in the dermis, below the lamina densa [16,17,18]. While collagen VII-specific autoantibodies are present in virtually all EBA patients, their exact prevalence in patients with IBD or other autoimmune bullous diseases is still controversial [23,24] or unknown, respectively
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