title: In vitro evolution of an antibody fragment population to find high-affinity hapten binders.
abstract: Recently, we constructed a focused antibody library tailored to interact with haptens. High functionality of this library was demonstrated, as specific binders could be retrieved to a range of different haptens. In the current study we have developed a mutagenesis and selection strategy in order to further fine-tune the hapten binding properties of these antibody fragments. Testosterone was chosen as model antigen for the investigation. A population, rather than a single clone, originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen concentration and having, or not having, soluble hapten present during the phage capture and elution steps. The different selection procedures resulted in a considerable increase in apparent affinities for several of the selected populations, from which the highest affinity antibody isolated had a K(D) of 2 nM, corresponding to an approximately 200-fold affinity improvement compared with the best clone of the starting population. Importantly, the polyclonal nature of the starting material allowed for the identification of novel unrelated variants that differed in fine-specificity, demonstrating that this approach is valuable for exploring different parts of structure space.
authors.affiliation: Department of Immunotechnology, Lund University, BMC D13 SE-221 84, Lund.
authors.forename: Helena
authors.initials: H
authors.lastname: Persson
authors.forename: Henrik
authors.initials: H
authors.lastname: Wallmark
authors.forename: Anne
authors.initials: A
authors.lastname: Ljungars
authors.forename: Johan
authors.initials: J
authors.lastname: Hallborn
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
mesh_terms: D000915:Antibody Affinity; D019020:Directed Molecular Evolution; D006241:Haptens / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D019151:Peptide Library; D013739:Testosterone / Q000276:immunology
chemical_list: D006241:Haptens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D013739:Testosterone