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Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, ribosome display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for ribosome display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte ribosome display method to isolate variants of V(H) antibody fragments with improved affinities.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.