title: High-Throughput IgG Conversion of Phage Displayed Fab Antibody Fragments by AmplYFast.
abstract: Phage display of antibody libraries is an invaluable strategy in antibody discovery. Many synthetic antibody library formats utilize monovalent antibody binding fragments (Fab), displayed on filamentous phage and expressed in Escherichia coli for selection and screening procedures, respectively. For most therapeutic applications, however, the final antibody candidate favors a bivalent immunoglobulin G (IgG) format, due to its particular effector function, half-life, and avidity.Here, we present an optimized subcloning method, termed AmplYFast, for the fast and convenient conversion of phage-displayed monovalent Fab fragments into full-length IgG or immunoglobulins of any other isotype. By using biotinylated primers, unique mammalian expression vectors, and multi-well plates, AmplYFast combines the rapid amplification, digestion, and ligation of recombinant Ig heavy and light chain sequences in an easy-to-operate high-throughput manner. Thus, AmplYFast improves quality and efficiency in DNA cloning and significantly minimizes timelines to antibody lead identification.
authors.affiliation: MorphoSys AG, Semmelweisstrasse 7, 82152, Planegg, Germany. andrea.sterner@morphosys.com.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sterner
authors.affiliation: MorphoSys AG, Semmelweisstrasse 7, 82152, Planegg, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Zehetmeier
mesh_terms: D001710:Biotin / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006207:Half-Life; D057166:High-Throughput Screening Assays; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D015342:DNA Probes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011994:Recombinant Proteins; D001710:Biotin
keywords: Biotinylated primer; DNA cloning; Fab; IgG; Immunoglobulin; Phage display; Recombinant antibody