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Antibody Affinity and Stability Maturation by Error-Prone PCR

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Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2702))

Abstract

Human antibodies are the most important class of biologicals, and antibodies – human and nonhuman – are indispensable as research agents and for diagnostic assays. When generating antibodies, they sometimes show the desired specificity profile but lack sufficient affinity for the desired application. In this article, a phage display-based method and protocol to increase the affinity of recombinant antibody fragments is given.

The given protocol starts with the construction of a mutated antibody gene library by error-prone PCR. Subsequently, the selection of high-affinity variants is performed by panning on immobilized antigen with washing conditions optimized for off-rate-dependent selection. A screening ELISA protocol to identify antibodies with improved affinity and an additional protocol to select antibodies with improved thermal stability is described.

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Acknowledgments

This review is an updated and revised version with improved protocols of [24].

Author information

Authors and Affiliations

  1. Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany

    Nora Langreder, Dorina Schäckermann, Tobias Unkauf, Maren Schubert, Federico Bertoglio & Michael Hust

  2. Wirtschaftsgenossenschaft deutscher Tierärzte eG (WDT), Garbsen, Germany

    Dorina Schäckermann

  3. Bayer Consumer Care AG, Basel, Switzerland

    Tobias Unkauf

  4. YUMAB GmbH, Science Campus Braunschweig-Süd, Braunschweig, Germany

    André Frenzel

  5. Choose Life Biotech SA, Bellinzona, Switzerland

    Federico Bertoglio

Authors
  1. Nora Langreder
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  2. Dorina Schäckermann
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  3. Tobias Unkauf
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  4. Maren Schubert
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  5. André Frenzel
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  6. Federico Bertoglio
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  7. Michael Hust
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Corresponding author

Correspondence to Michael Hust .

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Editors and Affiliations

  1. Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Langreder, N. et al. (2023). Antibody Affinity and Stability Maturation by Error-Prone PCR. In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_20

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  • DOI: https://doi.org/10.1007/978-1-0716-3381-6_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3380-9

  • Online ISBN: 978-1-0716-3381-6

  • eBook Packages: Springer Protocols

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