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Journal of the American Academy of Dermatology
Volume 68, n° 3
pages e89-e95 (mars 2013)
Doi : 10.1016/j.jaad.2011.12.032
accepted : 24 December 2011
JAAD Online

Sensitive and specific assays for routine serological diagnosis of epidermolysis bullosa acquisita
 

Lars Komorowski, PhD a, Ralf Müller, PhD b, Artem Vorobyev, MD b, Christian Probst, PhD a, Andreas Recke, MD b, Marcel F. Jonkman, MD, PhD c, Takashi Hashimoto, MD d, Soo-Chan Kim, MD e, Richard Groves, MD, FRCP f, Ralf J. Ludwig, MD b, Detlef Zillikens, MD b, Winfried Stöcker, MD a, Enno Schmidt, MD, PhD b, g,
a Institute of Experimental Immunology, EUROIMMUN AG, Luebeck, Germany 
b Department of Dermatology, University of Luebeck, Luebeck, Germany 
g Comprehensive Center for Inflammation Medicine, University of Luebeck, Luebeck, Germany 
c Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 
d Department of Dermatology, Kurume University School of Medicine and Kurume University Institute of Cutaneous Cell Biology, Kurume, Japan 
e Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Gangnam Severance Hospital, Seoul, Korea 
f Department of Immunodermatology, St John’s Institute of Dermatology, St Thomas’ Hospital, London, United Kingdom 

Correspondence to: Enno Schmidt, MD, PhD, Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Abstract
Background

Epidermolysis bullosa acquisita (EBA) is a severe autoimmune subepidermal blistering disease characterized by autoantibodies against the N-terminal collagenous domain (NC1) of type VII collagen (Col VII).

Objective

Development of reliable assays for the detection of anti-Col VII-NC1 antibodies.

Methods

NC1 was expressed in human HEK293 cells and used as target antigen in an enzyme-linked immunosorbent assay (ELISA) and in an immunofluorescence assay (IFA). These two assays were probed in a large cohort of patients with EBA (n = 73), bullous pemphigoid (BP, n = 72), anti-p200 pemphigoid (n = 24), anti-laminin 332 mucous membrane pemphigoid (MMP, n = 15), pemphigus vulgaris (PV, n = 24), and healthy control subjects (n = 254).

Results

The cut-off for the ELISA was optimized for accuracy by receiver-operating characteristics (area under the curve [AUC] = 0.9952). IgG reactivity against NC1 was detected in 69 of 73 EBA (94.5%) and 5 control sera (2 healthy controls and 3 BP patients), resulting in a specificity of 98.7%. The IFA showed a sensitivity of 91.8% and specificity of 99.8%. Reproducibility of the ELISA was demonstrated by an intra-class correlation coefficient of 0.97. IgG subclass analyses by ELISA revealed IgG1, IgG2, IgG3, and IgG4 anti-NC1 reactivity in 83.6%, 85.3%, 37.7%, and 83.6% of EBA sera, respectively.

Limitations

The novel assays were not evaluated prospectively and their use in monitoring serum levels during the disease course was not tested.

Conclusion

The two assays are highly specific and sensitive to diagnose EBA. Their diagnostic competence was demonstrated in a large cohort of well-characterized EBA sera.

The full text of this article is available in PDF format.

Key words : autoantibody, ELISA, immunofluorescence, type VII collagen

Abbreviations used : BP, Col VII, DEJ, EBA, ELISA, IF, NC1, PBS



 Drs Komorowski and Müller contributed equally and are listed in alphabetical order.
 Funding sources: This work was supported by the Schleswig-Holstein Cluster of Excellence in Inflammation Research (DFG EXC 306/1) to E.S., D.Z., and R.L and the Graduiertenkolleg “Modulation of Autoimmunity” (GRK 1727/1) to A.V.
 Disclosure: Drs Probst and Stõcker are employees and shareholders of EUROIMMUN AG. Dr Komorowski is an employee of EUROIMMUN AG.



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