Nucleic Acids Research · 1997 · 267 citations · 11 references
PharmacyBiotechnologyMolecular BiologyAntibody EngineeringDirected EvolutionMonoclonal AntibodiesAntibody DiscoveryAntibody Library ConstructionAntibody ScreeningAntibody Specificity EngineeringAntibody-antigen RecognitionDisplay Technologies For Antibody SelectionAntibody Library DesignAntigen Binding SelectionEnrichmentHigh-throughput Library SelectionLibrary-based Antibody SelectionManipulationMutant LibrariesPeptide Selection By Ribosome Display
We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.
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The "megaprimer" method of site-directed mutagenesis.
Gobinda Sarkar, S S Sommer · PubMed · 1990 · 1.2K citations