title: Improving the affinity and the fine specificity of an anti-cortisol antibody by parsimonious mutagenesis and phage display.
abstract: Immunoassays are widely used to determine steroid concentrations. However, they are limited by the specificity of antisteroid mAbs. We used the phage display system combined with molecular modeling and site-specific randomization to improve the affinity and the fine specificity of an anti-cortisol mAb. Using parsimonious mutagenesis, we have generated a library of mutant Ab fragments (scFv) derived from this Ab by randomizing five amino acids chosen by molecular modeling and Ab-hapten contact structural analysis. Anti-cortisol Ab fragments were selected from the library in the presence of steroid analogues to block cross-reacting binders. Specific elution with free cortisol allowed the recovery of clones with up to eightfold better affinity and fivefold less cross-reactivity than the wild-type scFv. This approach can be applied to any anti-hapten Ab and represents a useful approach for obtaining highly specific Abs for use in steroid immunoassays.
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, Marseille, France.
authors.forename: P
authors.initials: P
authors.lastname: Chames
authors.forename: S
authors.initials: S
authors.lastname: Coulon
authors.forename: D
authors.initials: D
authors.lastname: Baty
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000915:Antibody Affinity; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics* / Q000276:immunology; D001667:Binding, Competitive; D003001:Cloning, Molecular; D006854:Hydrocortisone / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000276:immunology*; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D006854:Hydrocortisone