Establishment of enzyme-linked immunosorbent assay for quantification of orotate phosphoribosyltransferase in gastric carcinoma

Yoichi Sakurai, Kazuki Sakamoto, Yoshikazu Sugimoto, Ikuo Yoshida, Toshihiko Masui, Shuhei Tonomura, Kazuki Inaba, Mitsutaka Shoji, Yasuko Nakamura, Ichiro Uyama, Yoshiyuki Komori, Masahiro Ochiai, Shiro Matsuura, Hideyuki Tanaka, Toshinori Oka, Masakazu Fukushima

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Abstract

A number of enzymes have been shown to be involved in the process of activation and/or degradation of 5-fluorouracil, and they are potential candidates for predicting factors of chemosensitivity to 5-fluorouracil. Among them, orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first-step activation process of 5-fluorouracil and therefore it has been shown to be an important enzyme for the prediction of sensitivity to 5-fluorouracil and its related derivatives. We developed a new enzyme-linked immunosorbent assay (ELISA) system to accurately assess intratumoral activity of orotate phosphoribosyltransferase. A new sandwich ELISA system was established using anti-OPRT polyclonal antibodies obtained from the rabbit immunized with the recombinant human peptides of the OPRT molecule. OPRT levels were measured in 8 human cancer xenografts transplanted in nude mice and 58 gastric cancer tissues using both a newly established ELISA and a conventional enzyme assay using radiolabeled 5-fluorouracil as a substrate. OPRT levels in 8 human cancer xenografts measured by this ELISA were significantly correlated with the OPRT enzyme activities (r2=0.782). Furthermore, OPRT activities measured in 58 gastric cancer tissues by enzyme assay were significantly correlated with those measured by the newly-established ELISA (r2=0.664). The ELISA system developed for the measurement of OPRT required a minimal amount of carcinoma tissue samples, which could be an easy-of-use assay system to predict sensitivity to 5-fluorouracil in gastric carcinoma. These results suggest that this newly-developed sandwich ELISA system for the quantification of OPRT is technically simple, feasible, and may be a useful tool to predict sensitivity to fluoropyrimidine-based anticancer chemotherapy in patients with gastric carcinoma and other cancers.

Original languageEnglish
Pages (from-to)1017-1022
Number of pages6
JournalGan to kagaku ryoho. Cancer & chemotherapy
Volume32
Issue number7
Publication statusPublished - 2005 Jul
Externally publishedYes

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ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Sakurai, Y., Sakamoto, K., Sugimoto, Y., Yoshida, I., Masui, T., Tonomura, S., Inaba, K., Shoji, M., Nakamura, Y., Uyama, I., Komori, Y., Ochiai, M., Matsuura, S., Tanaka, H., Oka, T., & Fukushima, M. (2005). Establishment of enzyme-linked immunosorbent assay for quantification of orotate phosphoribosyltransferase in gastric carcinoma. Gan to kagaku ryoho. Cancer & chemotherapy, 32(7), 1017-1022.