Affinity maturation by phage display.
Antibodies are indispensable tools for research, diagnostics, and therapy. However, sometimes antibodies with the most favourable specificity profile lack sufficient affinity for a desired application. Here, we describe a method to increase the affinity of recombinant scFv antibody fragments based on random mutagenesis and phage display under stringent conditions. Random mutations are inserted by performing several rounds of error-prone PCR. After construction of a mutated antibody gene library, affinity selection is performed by panning with washing conditions optimized for off-rate-dependent selection. Alternatively, panning in solution with competition can be used to enrich binders with improved binding properties.
Thie
Holger
H
Institute of Biochemistry and Biotechnology, Technical University Braunschweig, Braunschweig, Germany.
Voedisch
Bernd
B
Dübel
Stefan
S
Hust
Michael
M
Schirrmann
Thomas
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000915:Antibody Affinity / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012995:Solubility; D012996:Solutions; D017186:Titrimetry
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D012996:Solutions