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Abstract
Human antibodies are the most important class of biologicals, and antibodies - human and nonhuman - are indispensable as research agents and for diagnostic assays. When generating antibodies, they sometimes show the desired specificity profile but lack sufficient affinity for the desired application. In this article, a phage display-based method and protocol to increase the affinity of recombinant antibody fragments is given.The given protocol starts with the construction of a mutated antibody gene library by error-prone PCR. Subsequently, the selection of high-affinity variants is performed by panning on immobilized antigen with washing conditions optimized for off-rate-dependent selection. A screening ELISA protocol to identify antibodies with improved affinity and an additional protocol to select antibodies with improved thermal stability is described.
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