Use of micro-emulsion technology for the directed evolution of antibodies

Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2012-09, Vol.58 (1), p.28-33
Main Authors: Buhr, Diane L., Acca, Felicity E., Holland, Erika G., Johnson, Katie, Maksymiuk, Gail M., Vaill, Ada, Kay, Brian K., Weitz, David A., Weiner, Michael P., Kiss, Margaret M.
Format: Article
Language:English
Subjects:
Affinity maturation
Antibody Affinity
Antibody engineering
Antibody evolution
Bacteriophage M13 - genetics
Cell Surface Display Techniques
Directed Molecular Evolution
Emulsions
Enzyme-Linked Immunosorbent Assay
Escherichia coli - genetics
Humans
Kinetics
Mutagenesis
Peptide Library
Phage display
Phage ESCape
Polymerase Chain Reaction
Protein Binding
Protein Engineering
Protein Stability
Single-Chain Antibodies - biosynthesis
Single-Chain Antibodies - chemistry
Single-Chain Antibodies - genetics
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Summary:Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2012.07.007