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Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting

  • Anna Kaempffe
  • Sebastian Jäger
  • Doreen Könning
  • Harald Kolmar
  • Christian SchröterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

Abstract

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Key words

Fluorescence-activated cell sorting (FACS) Yeast surface display Fab display Species cross-reactivity Epitope binning Affinity Specificity B-cell receptor gene repertoires 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Anna Kaempffe
    • 1
    • 2
  • Sebastian Jäger
    • 1
    • 2
  • Doreen Könning
    • 2
  • Harald Kolmar
    • 1
  • Christian Schröter
    • 2
    Email author
  1. 1.Institute for Organic Chemistry and BiochemistryTechnische Universität DarmstadtDarmstadtGermany
  2. 2.Antibody-Drug Conjugates and Targeted NBE TherapeuticsMerck KGaADarmstadtGermany

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