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

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• 作者：Lars Komorowski ; PhD^a ; Ralf Mü ; ller ; 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 Stö ; cker ; MD^a ; Enno Schmidt ; MD ; PhD^b ; ^g ; ^{enno.schmidt@uk-sh.de}
• 关键词：autoantibody ; ELISA ; immunofluorescence ; type VII collagen
• 刊名：Journal of the American Academy of Dermatology
• 出版年：2013
• 出版时间：March, 2013
• 年：2013
• 卷：68
• 期：3
• 页码：e89-e95
• 全文大小：465 K

文摘

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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.