Changes in the specificity of antibodies against steroid antigens by introduction of mutations into complementarity-determining regions of the V(H) domain.
An attempt was made to change the specificity of antibodies (Abs) by introduction of mutations. A monoclonal Ab specific for 17alpha-hydroxyprogesterone (17-OHP) was used as the starting Ab. On the basis of a model that was generated by a computer-driven model-building system, we constructed a phage-display library of Abs in which 16 residues were mutated in three complementarity-determining regions of the heavy chain that appeared to form the steroid-binding pocket. We screened the library with 17-OHP and cortisol that had been conjugated with bovine serum albumin, and we isolated many clones that had retained 17-OHP-binding ability as well as clones with the newly developed ability to bind cortisol in addition to 17-OHP. We compared the amino acid sequences between 17-OHP-specific and cortisol-binding Abs, and then constructed several additional Abs. Our results indicated that a change in specificity could be achieved by changing only a single, critical amino acid residue. Models of the 17-OHP-and cortisol-binding pockets formed by the mutated Abs could explain these observations.
Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan.
Y
Y
Iba
N
N
Hayashi
J
J
Sawada
K
K
Titani
Y
Y
Kurosawa
D019326:17-alpha-Hydroxyprogesterone / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000918:Antibody Specificity / Q000235:genetics*; D000941:Antigens / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics*; D002460:Cell Line; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin; D006854:Hydrocortisone / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D019326:17-alpha-Hydroxyprogesterone; D006854:Hydrocortisone