Abstract:
:Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Kuwabara I,Maruyama H,Kamisue S,Shima M,Yoshioka A,Maruyama INdoi
10.1016/s0022-1759(99)00012-5keywords:
subject
Has Abstractpub_date
1999-04-22 00:00:00pages
89-99issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(99)00012-5journal_volume
224pub_type
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journal_title:Journal of immunological methods
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