Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast
<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme...
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| Format: | Article |
| Language: | English |
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BMC
2008-09-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/8/68 |
| _version_ | 1818178124942147584 |
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| author | Tombesi Marina Gellini Mara Ascione Alessandro Flego Michela Santoro Filippo Carpinelli Giulia Zamboni Silvia Mallano Alessandra Podo Franca Cianfriglia Maurizio |
| author_facet | Tombesi Marina Gellini Mara Ascione Alessandro Flego Michela Santoro Filippo Carpinelli Giulia Zamboni Silvia Mallano Alessandra Podo Franca Cianfriglia Maurizio |
| author_sort | Tombesi Marina |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p> |
| first_indexed | 2024-12-11T20:43:00Z |
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| id | doaj.art-3ef51113b1fe4dd6ad4e4a892668e1cc |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-11T20:43:00Z |
| publishDate | 2008-09-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-3ef51113b1fe4dd6ad4e4a892668e1cc2022-12-22T00:51:26ZengBMCBMC Biotechnology1472-67502008-09-01816810.1186/1472-6750-8-68Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from YeastTombesi MarinaGellini MaraAscione AlessandroFlego MichelaSantoro FilippoCarpinelli GiuliaZamboni SilviaMallano AlessandraPodo FrancaCianfriglia Maurizio<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p>http://www.biomedcentral.com/1472-6750/8/68 |
| spellingShingle | Tombesi Marina Gellini Mara Ascione Alessandro Flego Michela Santoro Filippo Carpinelli Giulia Zamboni Silvia Mallano Alessandra Podo Franca Cianfriglia Maurizio Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast BMC Biotechnology |
| title | Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast |
| title_full | Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast |
| title_fullStr | Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast |
| title_full_unstemmed | Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast |
| title_short | Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast |
| title_sort | generation and characterization of a human single chain fragment variable scfv antibody against cytosine deaminase from yeast |
| url | http://www.biomedcentral.com/1472-6750/8/68 |
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