title: Directing phage selections towards specific epitopes.
abstract: It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.
authors.affiliation: Cambridge Antibody Technology, Melbourn, UK.
authors.forename: H L
authors.initials: HL
authors.lastname: Parsons
authors.forename: J C
authors.initials: JC
authors.lastname: Earnshaw
authors.forename: J
authors.initials: J
authors.lastname: Wilton
authors.forename: K S
authors.initials: KS
authors.lastname: Johnson
authors.forename: P A
authors.initials: PA
authors.lastname: Schueler
authors.forename: W
authors.initials: W
authors.lastname: Mahoney
authors.forename: J
authors.initials: J
authors.lastname: McCafferty
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D001667:Binding, Competitive; D015374:Biosensing Techniques; D003001:Cloning, Molecular / Q000379:methods*; D003429:Cross Reactions; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005319:Fetal Hemoglobin / Q000276:immunology*; D006441:Hemoglobin A / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D012641:Selection, Genetic
chemical_list: D000939:Epitopes; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D006441:Hemoglobin A; D005319:Fetal Hemoglobin