title: Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid.
abstract: BACKGROUND
  Anti-epiligrin cicatricial pemphigoid (AECP) is a subepidermal blistering disease characterized by circulating anti-basement membrane autoantibodies to laminin 5.
  
  
  OBJECTIVE
  To evaluate the relative sensitivity of immunoblotting and immunoprecipitation techniques for the detection of anti-laminin 5 antibodies, comparative studies using reference laminin 5 antiserum as well as sera from patients with AECP, other immunobullous diseases, and normal volunteers were performed.
  
  
  METHODS
  Equivalent amounts of protein from five different substrates were studied by immunoblotting; immunoprecipitation experiments examined biosynthetically radiolabeled human keratinocyte (HK) extracts. Results HK extracellular matrix (ECM) was the most sensitive substrate for detection of antibodies to laminin 5; extracts of HKs, A-431 cells and HaCat cells represented alternative test substrates (though the later required higher amounts of protein input). Sera from patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. Immunoprecipitation studies found that a 10,000-fold dilution of reference laminin 5 antiserum retained the ability to identify laminin 5. Maximal dilutions of sera from AECP patients retaining the ability to immunoprecipitate laminin 5 ranged from 500 to 5,000.
  
  
  CONCLUSIONS
  Immunoprecipitation was the most sensitive technique for detection of anti-laminin 5 antibodies, while immunoblotting of HK ECM or HK extracts represented practical alternatives.
authors.affiliation: Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-4801, USA. zlazarov@mcw.edu
authors.forename: Zelmira
authors.initials: Z
authors.lastname: Lazarova
authors.forename: Cassian
authors.initials: C
authors.lastname: Sitaru
authors.forename: Detlef
authors.initials: D
authors.lastname: Zillikens
authors.forename: Kim B
authors.initials: KB
authors.lastname: Yancey
mesh_terms: D001323:Autoantibodies; D015815:Cell Adhesion Molecules / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D010391:Pemphigoid, Bullous / Q000276:immunology*; D015531:Radioimmunoprecipitation Assay; D000097794:Kalinin
chemical_list: D001323:Autoantibodies; D015815:Cell Adhesion Molecules; D000097794:Kalinin