title: [Establishment of enzyme-linked immunosorbent assay in the detection of BP180NC16A-specific IgE and its significance in bullous pemphigoid].
abstract: OBJECTIVE
  To establish a method of detecting circulating immunoglobulin E (IgE) autoantibodies for BP180NC16A and evaluate its significance in bullous pemphigoid (BP).
  
  
  METHODS
  GST-NC16A fusion proteins were expressed in Escherichia coli using the pGEX-2T expression system and purified by glutathione affinity chromatography.For optimal working conditions of enzyme-linked immunoabsorbent assay (ELISA), checkerboard titrations were performed with serial dilutions of antigen. Also optimized dilution of secondary antibody was confirmed. Sera samples from 56 patients with BP, 24 healthy controls, 18 with pemphigus and 1 with Stevens-Johnson syndrome at our hospital during February 2011 to October 2012 were examined by the modified ELISA approach. The optimal cut-off point for a positive result was selected with receiver operating characteristic analysis.
  
  
  RESULTS
  The optimized ELISA was performed with plates coated with 500 µg/ml GST-NC16A. And the optimal dilutions of sera samples and secondary antibody were 1: 10 and 1: 1000 respectively. According to the established cut-off value (0.549), 40 of 56 BP patients and none of controls had detectable levels of BP180NC16A IgE.
  
  
  CONCLUSIONS
  The established ELISA provides a highly specific tool for the detection of IgE anti-BP180NC16A in BP patients.
authors.affiliation: Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Ya-gang
authors.initials: YG
authors.lastname: Zuo
authors.forename: Xi-ping
authors.initials: XP
authors.lastname: Zhou
authors.forename: Chun-xia
authors.initials: CX
authors.lastname: He
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Duerna
authors.initials: D
authors.lastname: Tie
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Qiu-ning
authors.initials: QN
authors.lastname: Sun
mesh_terms: D001324:Autoantigens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D024041:Non-Fibrillar Collagens / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D000097805:Collagen Type XVII
chemical_list: D001324:Autoantigens; D007073:Immunoglobulin E; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII