title: 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.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Marwah Adly
authors.initials: MA
authors.lastname: Saleh
authors.forename: Ken
authors.initials: K
authors.lastname: Ishii
authors.forename: Yool-Ja
authors.initials: YJ
authors.lastname: Kim
authors.forename: Akihiro
authors.initials: A
authors.lastname: Murakami
authors.forename: Norito
authors.initials: N
authors.lastname: Ishii
authors.forename: Takashi
authors.initials: T
authors.lastname: Hashimoto
authors.forename: Enno
authors.initials: E
authors.lastname: Schmidt
authors.forename: Detlef
authors.initials: D
authors.lastname: Zillikens
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shirakata
authors.forename: Koji
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kitajima
authors.forename: Masayuki
authors.initials: M
authors.lastname: Amagai
mesh_terms: D001323:Autoantibodies / Q000737:chemistry; D001327:Autoimmune Diseases / Q000378:metabolism; D016022:Case-Control Studies; D024143:Collagen Type VII / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016107:Epidermolysis Bullosa Acquisita / Q000175:diagnosis* / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D017434:Protein Structure, Tertiary; D012372:ROC Curve; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D001323:Autoantibodies; D024143:Collagen Type VII; D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins