Prevalence of collagen VII-specific autoantibodies in patients with autoimmune and inflammatory diseases

Abstract

Background: Autoimmunity 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.

Methods: Based 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).

Results: By 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.

Conclusions: Using 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.

Figure 1

In silico analysis of B cell epitopes on human collagen VII. Linear and conformational antigenic determinants of collagen VII were analyzed in silico using BepiPred (http://www.cbs.dtu.dk/services/BepiPred/), CBTOPE (http://www.imtech.res.in/raghava/cbtope) and the Predictor component of Epitope Toolkit (EpiT; http://ailab.cs.iastate.edu/bcpreds/). The antigenic scores are plotted for the entire sequence of human collagen VII. The different regions of the autoantigen, including its non-collagenous (NC) 1 and 2 domains as well as the triple helical (TH) and hinge regions are shown in the lower part of the figure.

Figure 2

Recombinant forms of collagen VII used in this study. (a) Schematic representation of human collagen VII consisting of a central collagenous domain flanked by a large, 145 kDa N-terminal non-collagenous domain and a smaller, 30 kDa non-collagen domain at its C-terminus. The collagenous domain is interrupted by a 39 amino acid non-collagenous hinge region. The recombinant forms of collagen VII generated in this study are two N-terminally 8xhistidine tagged chimeric proteins termed His-hCVII-NC1-NC2 and His-hCVII-NC1-H-NC2 corresponding to the fused NC1 and NC2 domains (aa 1-1278, 2776-2944) and to the fused NC1, hinge and NC2 regions (1-1278, 1940-1979, 2776-2944) of the antigen, respectively. (b) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified recombinant His-hCVII-NC1-NC2 and His-hCVII-NC1-H-NC2 proteins shows their migration at around 153 (lane 2) and 158 kDa (lane 3), respectively. Weight markers of 200, 116, 97, 66 and 45 kDa are shown in lane 1. (c) Immunoblot analysis of the two recombinant proteins His-hCVII-NC1-NC2 (lanes 1 and 4) and His-hCVII-NC1-H-NC2 (lanes 2 and 5) as well as a recombinant form of collagen XVII [47] (lanes 3 and 6) using a monoclonal antibody specific to the NC1 domain of collagen VII (clone LH7.2; lanes 1-3) and a monoclonal antibody specific to soluble ectodomain of collagen XVII [47] (lanes 4-6).

Figure 3

Immunoreactivity of epidermolysis bullosa acquisita (EBA) autoantibodies with the recombinant forms of collagen VII. Purified, recombinant His-hCVII-NC1-NC2 (lanes 1-4) and His-hCVII-NC1-H-NC2 (lanes 5-8) were electrophoretically separated by 8% SDS-PAGE, transferred to nitrocellulose and immunoblotted with EBA patient's sera (lanes 1-3 and 5-7) and normal human sera (NHS) (lanes 4 and 8).

Figure 4

Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. Test performed with sera from patients with epidermolysis bullosa acquisita (n = 50) and controls (n = 160).

Figure 5

ELISA reactivity of human sera with the recombinant NC1-hinge-NC2 noncollagenous domains of collagen VII. Scatter plots represent optical density measurements of serum reactivity of epidermolysis bullosa acquisita (EBA), bullous pemphigoid (BP), pemphigus vulgaris (PV), Crohn's disease (CD), ulcerative colitis (UC) patients and healthy donors (NHS) with the purified recombinant chimeric collagen VII_. (His-hCVII-NC1-H-NC2). The cut-off of the assay is represented by a dotted line.

Figure 6

Autoantibodies against collagen VII are mainly of IgG4 isotype. Box-and-whiskers graphs represent descriptive summaries of the measurements for IgG1, IgG2, IgG3 and IgG4 autoantibodies against collagen VII by ELISA as described in Methods.