Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients

Marwah Adly Saleh, Ken Ishii, Yool Ja Kim, Akihiro Murakami, Norito Ishii, Takashi Hashimoto, Enno Schmidt, Detlef Zillikens, Yuji Shirakata, Koji Hashimoto, Yasuo Kitajima, Masayuki Amagai

Research output: Contribution to journalArticle

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Abstract

Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1. +. NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.

Original languageEnglish
Pages (from-to)169-175
Number of pages7
JournalJournal of Dermatological Science
Volume62
Issue number3
DOIs
Publication statusPublished - 2011 Jun

Fingerprint

Epidermolysis Bullosa Acquisita
Collagen Type VII
Autoantibodies
Epitopes
Assays
Enzyme-Linked Immunosorbent Assay
Serum
Antibodies
Bullous Pemphigoid
Pemphigus
Autoimmune Diseases
Sensitivity and Specificity

Keywords

  • Autoantibodies
  • ELISA
  • Epidermolysis bullosa acquisita
  • Type VII collagen

ASJC Scopus subject areas

  • Dermatology
  • Biochemistry
  • Molecular Biology

Cite this

Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients. / Saleh, Marwah Adly; Ishii, Ken; Kim, Yool Ja; Murakami, Akihiro; Ishii, Norito; Hashimoto, Takashi; Schmidt, Enno; Zillikens, Detlef; Shirakata, Yuji; Hashimoto, Koji; Kitajima, Yasuo; Amagai, Masayuki.

In: Journal of Dermatological Science, Vol. 62, No. 3, 06.2011, p. 169-175.

Research output: Contribution to journalArticle

Saleh, MA, Ishii, K, Kim, YJ, Murakami, A, Ishii, N, Hashimoto, T, Schmidt, E, Zillikens, D, Shirakata, Y, Hashimoto, K, Kitajima, Y & Amagai, M 2011, 'Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients', Journal of Dermatological Science, vol. 62, no. 3, pp. 169-175. https://doi.org/10.1016/j.jdermsci.2011.03.003
Saleh, Marwah Adly ; Ishii, Ken ; Kim, Yool Ja ; Murakami, Akihiro ; Ishii, Norito ; Hashimoto, Takashi ; Schmidt, Enno ; Zillikens, Detlef ; Shirakata, Yuji ; Hashimoto, Koji ; Kitajima, Yasuo ; Amagai, Masayuki. / Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients. In: Journal of Dermatological Science. 2011 ; Vol. 62, No. 3. pp. 169-175.
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abstract = "Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5{\%}) reacted with NC1 domain only, 7 sera (14.2{\%}) reacted with both NC1 and NC2 domains, and one serum (2{\%}) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8{\%} sensitivity and 98.1{\%} specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1. +. NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.",
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T1 - Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients

AU - Saleh, Marwah Adly

AU - Ishii, Ken

AU - Kim, Yool Ja

AU - Murakami, Akihiro

AU - Ishii, Norito

AU - Hashimoto, Takashi

AU - Schmidt, Enno

AU - Zillikens, Detlef

AU - Shirakata, Yuji

AU - Hashimoto, Koji

AU - Kitajima, Yasuo

AU - Amagai, Masayuki

PY - 2011/6

Y1 - 2011/6

N2 - Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1. +. NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.

AB - Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1. +. NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.

KW - Autoantibodies

KW - ELISA

KW - Epidermolysis bullosa acquisita

KW - Type VII collagen

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