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A method for the generation of combinatorial antibody libraries using pIX phage display
Changshou Gao1, Shenlan Mao, Gunnar Kaufmann
1Department of Chemistry, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Summary
This study developed a large human single-chain variable fragment (scFv) library using phage display technology. The library demonstrated high efficiency in selecting high-affinity and specific antibodies against diverse protein antigens.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Phage displayed combinatorial antibody libraries are established tools for antibody generation.
- Previous work demonstrated the utility of phage coat proteins pVII and pIX for displaying antibody Fv regions.
Purpose of the Study:
- To construct and characterize a large human single-chain Fv (scFv) library using the pIX phage display system.
- To evaluate the library's diversity, quality, and efficacy in selecting high-affinity and specific antibodies.
Main Methods:
- Construction of a 4.5 x 10^9 member human scFv library displayed on pIX of filamentous bacteriophage.
- Selection of scFv clones against six different protein antigens using panning.
- Affinity and specificity analysis of selected scFv clones using BIAcore kinetic analysis.
Main Results:
- Over 90% of selected clones showed positive antigen binding after three panning rounds.
- Selected scFvs exhibited high affinity, with dissociation constants in the nanomolar to subnanomolar range.
- High specificity was achieved, even between closely related proteins like RCA(60) and RCA(120).
Conclusions:
- The pIX-display scFv library is highly effective for generating antibodies with excellent affinity and specificity.
- This system shows potential to outperform pIII-display formats for antibody discovery.
- The developed library is well-suited for panning a broad range of target antigens.