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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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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).
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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).

Mentions: The immunoreactivity of the newly expressed recombinant chimeric forms of collagen VII was analyzed by immunoblotting using sera from reference EBA patients and healthy donors. Representatives examples are shown in Figure 3. IgG autoantibodies from EBA patients' sera (n = 5) recognized the recombinant forms His-hCVII-NC1-NC2 (Figure 3, lanes 1-3) and His-hCVII-NC1-H-NC2 (Figure 3, lanes 5-7) of collagen VII. Normal human sera (n = 2) did not react with these recombinant forms of collagen VII (Figure 3, lanes 4 and 8).


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)

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

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

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).
Mentions: The immunoreactivity of the newly expressed recombinant chimeric forms of collagen VII was analyzed by immunoblotting using sera from reference EBA patients and healthy donors. Representatives examples are shown in Figure 3. IgG autoantibodies from EBA patients' sera (n = 5) recognized the recombinant forms His-hCVII-NC1-NC2 (Figure 3, lanes 1-3) and His-hCVII-NC1-H-NC2 (Figure 3, lanes 5-7) of collagen VII. Normal human sera (n = 2) did not react with these recombinant forms of collagen VII (Figure 3, lanes 4 and 8).

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