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Related Experiment Videos

Antibody affinity maturation using bacterial surface display

P S Daugherty1, G Chen, M J Olsen

  • 1Department of Chemical Engineering, University of Texas, Austin 78712, USA.

Protein Engineering
|October 31, 1998
PubMed
Summary
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A novel system using E. coli surface display and fluorescence-activated cell sorting (FACS) efficiently screens antibody libraries. This method rapidly isolates high-affinity antibody variants, accelerating biopharmaceutical development.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • Screening antibody libraries is crucial for developing targeted therapeutics.
  • Existing methods for antibody library screening can be time-consuming and labor-intensive.
  • Developing rapid and efficient screening systems is essential for antibody discovery.

Purpose of the Study:

  • To develop and validate a quantitative system for screening single-chain variable fragment (scFv) antibody libraries.
  • To utilize Escherichia coli (E. coli) surface display and fluorescence-activated cell sorting (FACS) for high-affinity antibody isolation.
  • To demonstrate the system's capability in isolating both wild-type affinity and improved affinity antibody clones.

Main Methods:

  • Constructed combinatorial scFv libraries by randomizing antibody residues.

Related Experiment Videos

  • Employed E. coli surface display for antibody presentation.
  • Utilized FACS for quantitative screening and selection of high-affinity clones against a fluorescent hapten.
  • Confirmed antibody binding kinetics using surface plasmon resonance (SPR).

    • Main Results:

    • Successfully isolated antibody clones with wild-type and improved affinities (KD = 0.30+/-0.05 nM).
    • Identified a clone with a threefold lower dissociation constant than the wild-type antibody.
    • Demonstrated FACS as a rapid method for determining hapten dissociation rates without subcloning.
    • Validated FACS-based affinity measurements with SPR.

    Conclusions:

    • E. coli surface display coupled with FACS provides a powerful and rapid tool for isolating and characterizing desirable antibody mutants.
    • The developed system accelerates the discovery of high-affinity antibodies for therapeutic applications.
    • This approach offers significant potential for advancing antibody engineering and drug discovery pipelines.