Identification of IgG subclass and FVIII binding epitope of an acquired FVIII inhibitor in a bullous pemphigoid patient

Objective To identify the clinical and laboratory diagnosis of a bullous pemphigoid pa- tient with acquired hemophilia A(AH-A).To identify FⅧ binding epitope and IgG subclass of the FⅧ in- hibitor,and explore the molecular mechanism for AH-A pathogenesis.Methods Plasma FⅧ activity(FⅧ: C)was determined by one-stage assay,the titre of FⅧ inhibitor by Bethesda Unit(BU).IgG purification of patient plasma or normal pooled plasma was finished by protein A-agrose column chromatography.Activated partial thromboplastin time(APTT)was assayed for uncovering FⅧ inhibitor effect on FⅧ in vivo.Combined Western blot analysis by anti-IgG_1, IgG_2,IgG_3 and IgG_4 antibodies was used to determine the relative concen- tration of patient's IgG subclass.IgG subclass concentrations were quantified by nephelometric method.Sol- id-phase binding assay of FⅧ and FⅧ inhibitor,combined with Western blot was used to recognize the bind- ing epitope at which the FⅧ inhibitor bound to FⅧ.Results ①Plasma APTT value of patient was pro- longed evidently and could not be corrected by normal pooled plasma.Patient's FⅧ:C was1.5%.The titre of FⅧ inhibitor in patient plasma was 147.8 BU.②The purified patient IgG was able to inhibit FⅧ:C of normal pooled plasma significantly with a dose dependent mannar,and the patient plasma could prolong rabbil plasma APTT markedly with a time dependent manner.③The FⅧ inhibitor was predominantly then of IgG_4 subtype with a minority IgG_1,and the concentration of IgG_4 and IgG_1 in the patient was higher than that in normal.The FⅧ inhibitor reacted with FⅧ 44×10~3 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 44×10~3 fragment.The re- sults provides evidence for understanding the pathogenesis of AH-A.