An efficient strategy for cell-based antibody library selection using an integrated vector system
<p>Abstract</p> <p>Background</p> <p>Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we e...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2012-09-01
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| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1472-6750/12/62 |
| _version_ | 1811314863333441536 |
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| author | Yoon Hyerim Song Jin Ryu Chun Kim Yeon-Gu Lee Eun Kang Sunghyun Kim Sang |
| author_facet | Yoon Hyerim Song Jin Ryu Chun Kim Yeon-Gu Lee Eun Kang Sunghyun Kim Sang |
| author_sort | Yoon Hyerim |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.</p> <p>Results</p> <p>A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.</p> <p>Conclusions</p> <p>This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.</p> |
| first_indexed | 2024-04-13T11:19:59Z |
| format | Article |
| id | doaj.art-7e33bbba77394a4a97ef96e543cebf84 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T11:19:59Z |
| publishDate | 2012-09-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-7e33bbba77394a4a97ef96e543cebf842022-12-22T02:48:51ZengBMCBMC Biotechnology1472-67502012-09-011216210.1186/1472-6750-12-62An efficient strategy for cell-based antibody library selection using an integrated vector systemYoon HyerimSong JinRyu ChunKim Yeon-GuLee EunKang SunghyunKim Sang<p>Abstract</p> <p>Background</p> <p>Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.</p> <p>Results</p> <p>A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.</p> <p>Conclusions</p> <p>This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.</p>http://www.biomedcentral.com/1472-6750/12/62Phage displayAntibody libraryCD9Cell panningscFv-Fc |
| spellingShingle | Yoon Hyerim Song Jin Ryu Chun Kim Yeon-Gu Lee Eun Kang Sunghyun Kim Sang An efficient strategy for cell-based antibody library selection using an integrated vector system BMC Biotechnology Phage display Antibody library CD9 Cell panning scFv-Fc |
| title | An efficient strategy for cell-based antibody library selection using an integrated vector system |
| title_full | An efficient strategy for cell-based antibody library selection using an integrated vector system |
| title_fullStr | An efficient strategy for cell-based antibody library selection using an integrated vector system |
| title_full_unstemmed | An efficient strategy for cell-based antibody library selection using an integrated vector system |
| title_short | An efficient strategy for cell-based antibody library selection using an integrated vector system |
| title_sort | efficient strategy for cell based antibody library selection using an integrated vector system |
| topic | Phage display Antibody library CD9 Cell panning scFv-Fc |
| url | http://www.biomedcentral.com/1472-6750/12/62 |
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