title: [Identification of IgG subclass and FVIII binding epitope of an acquired FVIII inhibitor in a bullous pemphigoid patient].
abstract: OBJECTIVE
  To identify the clinical and laboratory diagnosis of a bullous pemphigoid patient with acquired hemophilia A (AH-A). To identify FVIII binding epitope and IgG subclass of the FVIII inhibitor, and explore the molecular mechanism for AH-A pathogenesis.
  
  
  METHODS
  Plasma FVIII activity( FVIII: C) was determined by one-stage assay, the titre of FYIII inhibitor by Bethesda Unit (BU). IgG purification of patient plasma or normal pooled plasma was finished by protein A-agarose column chromatography. Activated partial thromboplastin time (APTT) was assayed for uncovering FVIII inhibitor effect on FVIII in vivo. Combined Western blot analysis by anti-IgG1, IgG2, IgG3 and IgG4 antibodies was used to determine the relative concentration of patient' s IgG subclass. IgG subclass concentrations were quantified by nephelometric method. Solid-phase binding assay of FVIII and FVIII inhibitor, combined with Western blot was used to recognize the binding epitope at which the FVIII inhibitor bound to FVIII.
  
  
  RESULTS
  (1) Plasma APTT value of patient was prolonged evidently and could not be corrected by normal pooled plasma. Patient's FVIII: C was < 1.5%. The titre of FVIII inhibitor in patient plasma was 147.8 BU. (2) The purified patient IgG was able to inhibit FVIII: C of normal pooled plasma significantly with a dose dependent manner, and the patient plasma could prolong rabbit plasma APTT markedly with a time dependent manner. (3) The FVIII inhibitor was predominantly then of IgG4 subtype with a minority IgG1, and the concentration of IgG4 and IgG1 in the patient was higher than that in normal. The FVIII inhibitor reacted with FVIII 44 x 10(3) fragment epitope.
  
  
  CONCLUSIONS
  The inhibiting effect of FVIII inhibitors on FVIII: C in the bullous pemphigoid patient with AH-A is determined and the IgG subclass of the FVIII inhibitor is identified. A binding epitope for the FVIII inhibitor is a FVIII 44 x 10(3) fragment. The results provides evidence for understanding the pathogenesis of AH-A.
authors.affiliation: Department of Hematology and Molecular Hematology Laboratory, The Second Xiangya Hospital, Central South University, Changsha 410011, China.
authors.forename: Wen-Li
authors.initials: WL
authors.lastname: Zuo
authors.forename: Guang-Sen
authors.initials: GS
authors.lastname: Zhang
authors.forename: Zhi-Ju
authors.initials: ZJ
authors.lastname: Qing
authors.forename: Yun-Xiao
authors.initials: YX
authors.lastname: Xu
authors.forename: Li-Xin
authors.initials: LX
authors.lastname: Qin
authors.forename: Min
authors.initials: M
authors.lastname: Xu
mesh_terms: D000818:Animals; D000939:Epitopes; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000276:immunology; D005260:Female; D006467:Hemophilia A / Q000150:complications / Q000209:etiology / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D008875:Middle Aged; D010391:Pemphigoid, Bullous / Q000150:complications / Q000276:immunology*; D011817:Rabbits
chemical_list: D000939:Epitopes; D007074:Immunoglobulin G; D005169:Factor VIII