Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.
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.
Haack
K
K
Sektion Molekulare Diagnostik und Therapie, Chirurgische Universitätsklinik, Heidelberg, Germany.
Moebius
U
U
Knebel Doeberitz
M V
MV
Herfarth
C
C
Schackert
H K
HK
Gebert
J F
JF
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
D000911:Antibodies, Monoclonal; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase