title: Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.
abstract: The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
authors.forename: S A
authors.initials: SA
authors.lastname: Narang
authors.forename: D A
authors.initials: DA
authors.lastname: Brummell
authors.forename: V
authors.initials: V
authors.lastname: Sharma
authors.forename: D
authors.initials: D
authors.lastname: Bilous
authors.forename: G
authors.initials: G
authors.lastname: Dubuc
authors.forename: C R
authors.initials: CR
authors.lastname: MacKenzie
authors.forename: J
authors.initials: J
authors.lastname: Michniewicz
authors.forename: J
authors.initials: J
authors.lastname: Sadowska
authors.forename: N
authors.initials: N
authors.lastname: Anand
authors.forename: N M
authors.initials: NM
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D001666:Binding Sites, Antibody; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D019081:O Antigens; D009838:Oligodeoxyribonucleotides; D011135:Polysaccharides, Bacterial / Q000235:genetics* / Q000276:immunology; D012475:Salmonella / Q000235:genetics*
chemical_list: D000936:Antigen-Antibody Complex; D019081:O Antigens; D009838:Oligodeoxyribonucleotides; D011135:Polysaccharides, Bacterial