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Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Published on: January 17, 2015
Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen
S Barth1, U Weidenmüller, M K Tur
1Department of Veterinary Science, Institute of Immunology and Molecular Biology, Luisenstr. 56, University of Berlin, 10117, Germany. stefan.barth@uni-koeln.de
Abstract:
A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.
Insights
Identifying tumor antigens is challenging. This study shows that scFv phages can detect target antigens within a lambda phage cDNA expression library, aiding in cancer research.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Phage display is used to select single-chain variable fragment (scFv) antibodies against tumor cells.
- Identifying the specific antigen targeted by these scFv antibodies remains a significant challenge.
- Existing methods like ELISA and FACS evaluate binding but do not definitively identify the antigen.
Purpose of the Study:
- To demonstrate a method for detecting the target antigen of a preselected scFv displayed on phage.
- To validate antigen detection within a complex, immobilized lambda phage cDNA expression library.
Main Methods:
- Utilized scFv phage display libraries selected on tumor cells.
- Employed an immobilized lambda phage cDNA expression library containing both tumor-specific and irrelevant clones (including CD30-negative breast cancer cDNA and human CD30 receptor cDNA).
- Documented the interaction between anti-CD30 scFv phages and immobilized antigens via blotting on nitrocellulose filters.
Main Results:
- Successfully detected the CD30 antigen using anti-CD30 scFv phages in the lambda phage cDNA expression library.
- Demonstrated antigen detection is feasible with at least 5x10^12 plaque-forming units of filamentous phage per blot.
- Showed successful detection across different ratios of CD30 receptor to breast cancer specific clones (1:1 and 1:200).
Conclusions:
- Phage-displayed scFv can identify their target antigens within complex cDNA expression libraries.
- This technique provides a novel approach to antigen discovery in cancer research.
- Facilitates the characterization of tumor-associated antigens and the development of targeted therapies.
