title: Development of a novel ELISA system for detection of anti-BP180 IgG and characterization of autoantibody profile in bullous pemphigoid patients.
abstract: BACKGROUND
  The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection.
  
  
  OBJECTIVE
  Because BP180 autoantibody reactivity is not restricted to NC16A, we have investigated the possibility of developing an ELISA based on selected epitopes additional to this immunodominant region.
  
  
  METHODS
  Initially 78 BP sera were tested using an NC16A ELISA and IgG reactivity was detected in 64 BP sera (82%). The 14 NC16A-negative BP sera were then analysed by immunological screening against seven BP180-specific epitopes. Recombinant phages displaying BP180 epitopes were grown as plaques, blotted onto a nitrocellulose filter and incubated with BP sera.
  
  
  RESULTS
  Three and five NC16A-negative BP sera reacted with epitopes AA 1080-1107 and AA 1331-1404 of the BP180 ectodomain, respectively. Thus, a novel ELISA with GST-1080 and GST-1331 (GST-1080/1331) was developed: 32 of 78 BP sera (41%) proved positive by this assay. The combined use of ELISAs with GST-NC16A and GST-1080/1331 detected IgG reactivity in 72 of 78 BP sera, increasing the sensitivity from 82% to 92%. In addition, autoreactivity against the three extracellular epitopes appeared to be related to the presence of both skin and mucosal involvement as assessed by Fisher's exact probability test.
  
  
  CONCLUSIONS
  Our findings further characterize the autoimmune response in BP by identifying a subgroup of NC16A-negative patients who react with different BP180 extracellular epitopes. The developed ELISA system appears more sensitive than the ELISA based on NC16A alone and also informative about the epitope profile of BP patients.
authors.affiliation: Molecular and Cell Biology Laboratory, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Mariotti
authors.forename: F
authors.initials: F
authors.lastname: Grosso
authors.forename: M
authors.initials: M
authors.lastname: Terracina
authors.forename: M
authors.initials: M
authors.lastname: Ruffelli
authors.forename: P
authors.initials: P
authors.lastname: Cordiali-Fei
authors.forename: F
authors.initials: F
authors.lastname: Sera
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
authors.forename: A
authors.initials: A
authors.lastname: Mastrogiacomo
authors.forename: G
authors.initials: G
authors.lastname: Di Zenzo
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood*; D008875:Middle Aged; D009092:Mucous Membrane / Q000276:immunology / Q000473:pathology; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000175:diagnosis / Q000276:immunology* / Q000473:pathology; D012680:Sensitivity and Specificity; D000097805:Collagen Type XVII
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII