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

Licarete E, Ganz S, Recknagel MJ, Di Zenzo G, Hashimoto T, Hertl M, Zambruno G, Hundorfean G, Mudter J, Neurath MF, Bruckner-Tuderman L, Sitaru C - BMC Immunol. (2012)

Bottom Line: 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).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%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, University of Freiburg, Hauptstr, 7, Freiburg 79104, Germany.

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.

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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.
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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.

Mentions: 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. To further our understanding about the distribution of B cell epitopes on collagen VII, we applied in silico analysis of both linear and conformational epitopes using different algorithms. The results revealed that NC1 and NC2 domains as well as the hinge region within the triple helix of collagen VII contain more antigenic sites compared with its collagenous domain (Figure 1, Additional file 1: Table S1).


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

Licarete E, Ganz S, Recknagel MJ, Di Zenzo G, Hashimoto T, Hertl M, Zambruno G, Hundorfean G, Mudter J, Neurath MF, Bruckner-Tuderman L, Sitaru C - BMC Immunol. (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368718&req=5

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.
Mentions: 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. To further our understanding about the distribution of B cell epitopes on collagen VII, we applied in silico analysis of both linear and conformational epitopes using different algorithms. The results revealed that NC1 and NC2 domains as well as the hinge region within the triple helix of collagen VII contain more antigenic sites compared with its collagenous domain (Figure 1, Additional file 1: Table S1).

Bottom Line: 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).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%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, University of Freiburg, Hauptstr, 7, Freiburg 79104, Germany.

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.

Show MeSH
Related in: MedlinePlus