title: Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.
abstract: Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.
authors.affiliation: Sektion Molekulare Diagnostik und Therapie, Chirurgische Universitätsklinik, Heidelberg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Haack
authors.forename: U
authors.initials: U
authors.lastname: Moebius
authors.forename: M V
authors.initials: MV
authors.lastname: Knebel Doeberitz
authors.forename: C
authors.initials: C
authors.lastname: Herfarth
authors.forename: H K
authors.initials: HK
authors.lastname: Schackert
authors.forename: J F
authors.initials: JF
authors.lastname: Gebert
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D043525:Cytosine Deaminase; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D009700:Nucleoside Deaminases / Q000032:analysis* / Q000276:immunology; D011233:Precipitin Tests; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase