High-Throughput IgG Conversion of Phage Displayed Fab Antibody Fragments by AmplYFast

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

Abstract

Phage display of antibody libraries is an invaluable strategy in antibody discovery. Many synthetic antibody library formats utilize monovalent antibody binding fragments (Fab), displayed on filamentous phage and expressed in Escherichia coli for selection and screening procedures, respectively. For most therapeutic applications, however, the final antibody candidate favors a bivalent immunoglobulin G (IgG) format, due to its particular effector function, half-life, and avidity.

Here, we present an optimized subcloning method, termed AmplYFast, for the fast and convenient conversion of phage-displayed monovalent Fab fragments into full-length IgG or immunoglobulins of any other isotype. By using biotinylated primers, unique mammalian expression vectors, and multi-well plates, AmplYFast combines the rapid amplification, digestion, and ligation of recombinant Ig heavy and light chain sequences in an easy-to-operate high-throughput manner. Thus, AmplYFast improves quality and efficiency in DNA cloning and significantly minimizes timelines to antibody lead identification.

Key words

Immunoglobulin Phage display IgG Fab DNA cloning Biotinylated primer Recombinant antibody 

Notes

Acknowledgment

The authors like to thank Stephanie Patzelt and Christoph Erkel for their support and scientific discussions, as well as Thomas Tiller and Katja Siegers for comments on the manuscript.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.MorphoSys AGPlaneggGermany

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