Optimisation of antibody affinity by ribosome display using error-prone or site-directed mutagenesis

Affinity optimisation of antibodies can be achieved with great success by using directed evolution approaches, that is, the creation of and selection from diverse libraries. Here, we describe in detail methods to optimise antibody affinity for an antigen through directed evolution using ribosome display. Diversification of antibody single chain variable (scFv) domains is carried out by error-prone PCR and oligonucleotide-directed mutagenesis to generate random and targeted libraries respectively. Subsequent libraries are converted to ribosome display format and taken through cycles of transcription, translation, and selection. Since the starting point and the recovered product are linear DNA, this can easily be manipulated further to allow accumulation of beneficial mutations through iterative cycles of selection.