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

Autor:	Carrara SC; Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratory, Biologics Technology and Development, Darmstadt, Germany., Bogen JP; Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratory, Biologics Technology and Development, Darmstadt, Germany., Grzeschik J; Ferring Darmstadt Laboratory, Biologics Technology and Development, Darmstadt, Germany., Hock B; Ferring International Center S.A., Saint-Prex, Switzerland., Kolmar H; Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany. kolmar@biochemie-tud.de.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2491, pp. 177-193.
DOI:	10.1007/978-1-0716-2285-8_10
Abstrakt:	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. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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