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

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Yeast surface display (YSD) is a key screening method for isolating monoclonal antibodies (mAbs).
• Phage display is currently the gold standard for isolating mAbs against difficult targets like GPCRs and ion channels.
• Limitations exist in current methods for antibody discovery against complex membrane proteins.

Purpose of the Study:

• To establish and detail a protocol for isolating mAbs using YSD with FACS-assisted biopanning.
• To demonstrate the isolation of high-affinity antibodies binding antigens in their native cellular environment.
• To highlight essential characterization methods for antibodies generated via this YSD approach.

Main Methods:

• Development of a step-by-step protocol for YSD biopanning.
• Utilizing fluorescence-activated cell sorting (FACS) for enrichment of antibody-displaying yeast cells.
• Employing antigen-displaying mammalian cells to ensure correct antigen conformation and epitope accessibility.

Main Results:

• Successful isolation of diverse antibodies with high affinity for their respective antigens.
• Demonstration of antibody binding within the natural cellular context of the target antigen.
• Validation of the FACS-assisted YSD approach for antibody discovery.

Conclusions:

• FACS-assisted YSD provides a robust method for isolating high-affinity mAbs against challenging targets.
• The use of mammalian cells in YSD biopanning ensures antigen integrity and facilitates antibody discovery.
• This protocol offers a valuable alternative for antibody generation, particularly for membrane proteins.