Epitope analysis and utility of monoclonal antibodies to native and recombinant human thymidylate synthase.
The expression of thymidylate synthase (TS) in human cancer tissues has been suggested to be a prognostic factor for patients receiving 5-fluorouracil-based chemotherapy. We generated monoclonal antibodies to both recombinant and native TS and analyzed the epitopes on the TS molecule. Two monoclonal antibodies were obtained from recombinant human TS proteins (RTSMA1 and RTSMA2) and two monoclonal antibodies raised to native TS in human cancers (NTSMA1 and NTSMA2) were obtained, and were found to have a high affinity and specificity for TS proteins. To identify the human TS epitope that these monoclonal antibodies recognized, we constructed plasmids to produce full-length and two partially deleted TS proteins fused to glutathion-S-transferase (GST). Western blot analysis showed that RTSMA1 and 2 only reacted with full-length TS and NTSMA1 and 2 reacted with all three recombinant TS proteins, suggesting that the epitopes of the former were located at C-terminal sites (D186-V313), and those of the latter were at N-terminal sites (M1-M61). The RTSMA 1 and 2 epitopes were more accurately mapped using 8 oligopeptides to the region of I267 to L282 which was situated at the surface of the TS protein. Highly sensitive detection of human TS was also possible by sandwich ELISA using a combination of different types of antibody rather than a single type. In conclusion, different type monoclonal antibodies to human TS protein may contribute to the detection of TS in cancer patients.
Okabe
H
H
The Second Cancer Laboratory, Taiho Pharmaceutical Co., Ltd., Hanno-City, Saitama 357-8527, Japan.
Koizumi
K
K
Tsujimoto
H
H
Fukushima
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D015153:Blotting, Western; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013940:Thymidylate Synthase / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013940:Thymidylate Synthase