title: A general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition.
abstract: We report here a generally applicable method for the selective covalent attachment of a reporter molecule to a replicating entity that allows one to obtain specific binders from a single round of library screening. We show that selective biotinylation of phage particles displaying a binder to any given target can be achieved by application of a coupled enzyme reaction on the surface of the target-binding phage particles that includes a peroxidase, an oxidase and a catalase. Due to the covalent linkage of biotin together with the tight and stable interaction of biotin with streptavidin, very stringent wash conditions for removal of nonspecific binders can be applied. The method termed (3)CARD (triple catalytic reporter deposition) was successfully applied to single-round screening of a phage display library of camelid single-domain antibodies against three different target proteins.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, D-64287 Darmstadt, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Maaß
authors.forename: Tim
authors.initials: T
authors.lastname: Heiseler
authors.forename: Franziska
authors.initials: F
authors.lastname: Maaß
authors.forename: Janine
authors.initials: J
authors.lastname: Fritz
authors.forename: Thomas
authors.initials: T
authors.lastname: Hofmeyer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Glotzbach
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D019921:Biotinylation; D002161:Camelids, New World; D004800:Enzymes, Immobilized / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D019151:Peptide Library; D010504:Periodic Acid / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000276:immunology*
chemical_list: D000941:Antigens; D004800:Enzymes, Immobilized; D019151:Peptide Library; D061905:Single-Domain Antibodies; D010504:Periodic Acid; C009288:metaperiodate; D010088:Oxidoreductases; D009195:Peroxidase
keywords: antibody engineering; catalysed reporter deposition; oxidase; peroxidase; phage display screening