Selection of stable scFv antibodies by phage display.
ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl. Irreversible scFv denaturation, which is a prerequisite for efficient selection, is achieved by combining denaturation with reduction of the intradomain disulfide bonds. Repeated selection cycles of increasing stringency result in enrichment of stabilized scFv fragments. Procedures for constructing a randomly mutated scFv library by error-prone PCR and phage display selection for enrichment of stable scFv antibodies from the library are described here.
Department of Biotechnology, University of Turku, Turku, Finland. eechbr@utu.fi
Eeva-Christine
EC
Brockmann
D001435:Bacteriophages / Q000378:metabolism / Q000472:pathogenicity; D061505:Cell Surface Display Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007118:Immunoassay; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000235:genetics*; D014170:Transformation, Genetic / Q000187:drug effects
D057127:Single-Chain Antibodies; D019791:Guanidine