Protocol

Antibody Engineering

Volume 907 of the series Methods in Molecular Biology pp 281-292

Date:

Evolution of Antibodies In Vitro by Ribosome Display

  • Bryan M. EdwardsAffiliated withCrescendo Biologics Ltd.
  • , Mingyue HeAffiliated withThe Inositide Laboratory, The Babraham Institute Email author 

* Final gross prices may vary according to local VAT.

Get Access

Abstract

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 VH antibody fragments with improved affinities.

Key words

Ribosome display In situ RT-PCR Single-domain antibody Cell-free expression