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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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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.
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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.

Mentions: Applying the cut-off value of 0.322 defined by ROC analysis for the newly developed ELISA showed that 47 EBA (94%; 95% CI: 87%-100%; n = 50), 2 CD (4%; 95% CI: 0%-9.43%; n = 50), 8 UC (16%; 95% CI: 5.8%-26%; n = 50), 2 BP (2.63%; 95% CI: 0%-6.23%; n = 76), 4 PV (9.52%; 95% CI: 0%-18.4%; n = 42) patients and 4 of the healthy donors (1.63%; 95% CI: 0%-3.21%; n = 245) showed IgG reactivity against the chimeric NC1-hinge-NC2-hCVII protein (Figure 5). Therefore, a sensitivity and a specificity of 94% (95% CI: 83.4%-98.75%) and 98%(95% CI: 94%-100%), respectively, were calculated for the ELISA detecting collagen VII-specific IgG autoantibodies in patients with EBA. The area under the curve (AUC) was 0.984 (95% CI: 96.3%-100%) indicating an excellent discriminatory power. The accuracy of the ELISA using only the NC1-NC2 domains of collagen VII was only slightly lower as demonstrated by a sensitivity of 92% (95% CI: 80.7%-97.7%) and a specificity of 97.50% (95% CI: 93.7%-99.3%) with an AUC of 0.980 (data not shown). The immunoassays using the 2 recombinant forms of collagen VII correlated well regarding their capacity to detect specific autoantibodies (r = 0.95; p < 0.0001).


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)

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

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

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.
Mentions: Applying the cut-off value of 0.322 defined by ROC analysis for the newly developed ELISA showed that 47 EBA (94%; 95% CI: 87%-100%; n = 50), 2 CD (4%; 95% CI: 0%-9.43%; n = 50), 8 UC (16%; 95% CI: 5.8%-26%; n = 50), 2 BP (2.63%; 95% CI: 0%-6.23%; n = 76), 4 PV (9.52%; 95% CI: 0%-18.4%; n = 42) patients and 4 of the healthy donors (1.63%; 95% CI: 0%-3.21%; n = 245) showed IgG reactivity against the chimeric NC1-hinge-NC2-hCVII protein (Figure 5). Therefore, a sensitivity and a specificity of 94% (95% CI: 83.4%-98.75%) and 98%(95% CI: 94%-100%), respectively, were calculated for the ELISA detecting collagen VII-specific IgG autoantibodies in patients with EBA. The area under the curve (AUC) was 0.984 (95% CI: 96.3%-100%) indicating an excellent discriminatory power. The accuracy of the ELISA using only the NC1-NC2 domains of collagen VII was only slightly lower as demonstrated by a sensitivity of 92% (95% CI: 80.7%-97.7%) and a specificity of 97.50% (95% CI: 93.7%-99.3%) with an AUC of 0.980 (data not shown). The immunoassays using the 2 recombinant forms of collagen VII correlated well regarding their capacity to detect specific autoantibodies (r = 0.95; p < 0.0001).

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