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A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening
Ziyi Liu1,2, Dokyun Kim1,2, Seokmin Kang1,2
1Cancer Biology Department, Infection Biology Program, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Methods and Protocols
|February 23, 2024
Summary
Researchers developed a method to create a large library of single-chain variable fragments (scFv) from human patients. This library, screened using phage display, yields high-affinity antibodies for research and clinical use.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Monoclonal antibodies (mAbs) are crucial in research and medicine due to their specificity.
- Phage display technology enables in vitro screening of antibodies for high antigen affinity.
- Single-chain variable fragments (scFv) offer high binding affinity and a smaller size compared to traditional antibodies.
Purpose of the Study:
- To detail a procedure for constructing a diverse human scFv phagemid library.
- To outline a high-throughput screening protocol using phage display affinity selection.
- To provide a valuable resource for identifying high-affinity scFvs for research and clinical applications.
Main Methods:
- Construction of a scFv phagemid library using 348 primer combinations to ensure broad human antibody repertoire coverage and minimize bias.
- Generation of a scFv library exceeding 1 × 10^8 diversity.
- High-throughput screening of over 1200 scFv candidates using phage display and enzyme-linked immunosorbent assay (ELISA).
Main Results:
- Successful generation of a highly diverse human scFv phagemid library.
- Establishment of a robust phage display screening methodology.
- Identification of scFv candidates with high affinity for target antigens.
Conclusions:
- The developed protocol provides a comprehensive method for creating and screening scFv libraries.
- This approach is expected to accelerate the discovery of novel therapeutic and diagnostic antibodies.
- The generated library serves as a valuable resource for advancing antibody-based research and clinical applications.
