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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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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).
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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).

Mentions: The recombinant proteins were expressed in mammalian cells and purified by metallochelate affinity chromatography. When separated by SDS-PAGE, the recombinant collagen VII forms containing the NC1 fused with the NC2 domain (His-hCVII-NC1-NC2) as well as the hinge region (His-hCVII-NC1-H-NC2), migrated consistently with their calculated molecular masses of 153 kDa (Figure 2b, lane 2) and 158 kDa (Figure 2b, lane 3), respectively. A monoclonal antibody specific for the NC1 domain of collagen VII recognized both recombinant forms by immunoblot analysis (Figure 2c, lanes 1 and 2).


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)

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

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

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).
Mentions: The recombinant proteins were expressed in mammalian cells and purified by metallochelate affinity chromatography. When separated by SDS-PAGE, the recombinant collagen VII forms containing the NC1 fused with the NC2 domain (His-hCVII-NC1-NC2) as well as the hinge region (His-hCVII-NC1-H-NC2), migrated consistently with their calculated molecular masses of 153 kDa (Figure 2b, lane 2) and 158 kDa (Figure 2b, lane 3), respectively. A monoclonal antibody specific for the NC1 domain of collagen VII recognized both recombinant forms by immunoblot analysis (Figure 2c, lanes 1 and 2).

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