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. 2022:2491:177-193.
doi: 10.1007/978-1-0716-2285-8_10.

Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting

Stefania C Carrara #  1   2 , Jan P Bogen #  1   2 , Julius Grzeschik  2 , Björn Hock  3 , Harald Kolmar  4
Affiliations

Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting

Stefania C Carrara et al. Methods Mol Biol. 2022.

Abstract

Yeast surface display (YSD) emerged as a prominent screening methodology for the isolation of monoclonal antibodies (mAbs) against various antigens. However, phage display remains the gold standard in cell panning-based screenings to isolate mAbs against difficult-to-screen targets, such as G-protein coupled receptors (GPCR) and ion channels. Herein we describe a step-by-step protocol to establish and perform the isolation of mAbs using YSD in a fluorescence-activated cell sorting (FACS)-assisted biopanning manner, yielding a variety of antibodies binding their antigen with high affinity in the natural environment of the cell. Upon mixing antibody-displaying yeast cells with antigen-displaying mammalian cells, complexes are specifically formed and isolated for enrichment of yeast cells encoding binders against the antigen. The utilization of mammalian cells expressing the respective target accounts for accessibility of the epitope and the correct conformation of the antigen. Furthermore, critical characterization methods mandatory for this kind of antibodies are illuminated.

Keywords: Antibody library screening; FACS; Hit discovery; Yeast biopanning; Yeast surface display.

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