Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients.
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.
Kuwabara
I
I
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Maruyama
H
H
Kamisue
S
S
Shima
M
M
Yoshioka
A
A
Maruyama
I N
IN
D000042:Absorption; D010582:Bacteriophage lambda; D001483:Base Sequence; D003545:Cysteine; D004220:Disulfides; D018604:Epitope Mapping / Q000379:methods*; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015723:Gene Library; D005822:Genetic Vectors; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D004220:Disulfides; D018985:Epitopes, B-Lymphocyte; D007518:Isoantibodies; D005169:Factor VIII; D003545:Cysteine