title: False-negative results in immunoblot assay of serum IgA antibodies reactive with the 180-kDa bullous pemphigoid antigen: the importance of primary incubation temperature.
abstract: BACKGROUND
  Different subepidermal autoimmune blistering skin disorders are characterized by linear deposition of IgA, sometimes accompanied by linear IgG, along the epidermal basement membrane zone. Identification of the targeted autoantigen is usually attempted by immunoblotting. Although immunoblotting works well for human IgG, the method is less successful for IgA and often no or only faint signals are obtained.
  
  
  OBJECTIVE
  To improve the method of immunoblotting for diagnosis of IgA-mediated bullous dermatoses.
  
  
  METHODS
  Eleven sera, selected from patients with linear deposition of IgA along the epidermal basement membrane zone in vivo, were tested by immunoblotting for antigen specificity using different primary incubation temperatures.
  
  
  RESULTS
  No reliable information regarding IgA antigen specificity was obtained when the primary incubation was undertaken at room temperature. In 10 of 11 sera, IgA bound to the 180-kDa bullous pemphigoid antigen (BP180) when the primary incubation temperature was increased to 37 degrees C.
  
  
  CONCLUSIONS
  Primary incubation at room temperature may result in false-negative results in the IgA-BP180 immunoblot assay.
authors.affiliation: Centre for Blistering Skin Diseases, Department of Dermatology, Groningen University Hospital, The Netherlands. h.h.pas@derm.azg.nl
authors.forename: H H
authors.initials: HH
authors.lastname: Pas
authors.forename: G J
authors.initials: GJ
authors.lastname: Kloosterhuis
authors.forename: M C
authors.initials: MC
authors.lastname: de Jong
authors.forename: M F
authors.initials: MF
authors.lastname: Jonkman
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000276:immunology*; D001327:Autoimmune Diseases / Q000175:diagnosis*; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D005188:False Negative Reactions; D005260:Female; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007070:Immunoglobulin A / Q000097:blood* / Q000276:immunology; D008297:Male; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D012872:Skin Diseases, Vesiculobullous / Q000175:diagnosis*; D013696:Temperature; D000097805:Collagen Type XVII
chemical_list: D001324:Autoantigens; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D007070:Immunoglobulin A; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII; C486268:DST protein, human