Mapping of the minimal domain encoding a conformational epitope by λ phage surface display: factor VIII inhibitor antibodies from haemophilia A patients
| Autor: | Ichi N Maruyama, Seiki Kamisue, Ichiro Kuwabara, Hiroko Maruyama, Akira Yoshioka, Midori Shima |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities Phage display Protein Conformation Genetic Vectors Molecular Sequence Data Immunology Hemophilia A Epitope Absorption Bacteriophage Structure-Activity Relationship Protein structure Isoantibodies hemic and lymphatic diseases Humans Immunology and Allergy Cysteine Disulfides Binding site Gene Library Factor VIII Base Sequence biology Chemistry Sequence Analysis DNA Lambda phage biology.organism_classification Bacteriophage lambda Molecular biology Protein tertiary structure Biochemistry Epitopes B-Lymphocyte Epitope Mapping Conformational epitope |
| Zdroj: | Journal of Immunological Methods. 224:89-99 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/s0022-1759(99)00012-5 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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