Development, characterization and application of an antibody against 5-fluoro-2'deoxyuridine-5'monophosphate, the active metabolite of 5-fluorouracil

G. J. Peters, E. Laurensse, H. W M Steinbusch, J. De Vente, K. Smid, C. L. Van der Wilt, H. M. Pinedo

Research output: Contribution to journalArticle

Abstract

5-Fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is the active metabolite of the anticancer agent 5-fluorouracil (5FU). Antibodies against a conjugate of thyroglobulin with acetylated FdUMP were raised in 4 rabbits. The maximal titer was reached after 3-5 boosters. Subsequently the antisera were collected and partially purified. In a competition assay a 100-fold excess of the natural nucleotide dUMP could displace tritiated FdUMP (20 pmol per assay) by about 50% in all antisera. However tritiated dUMP itself did not bind to the antibody. No cross-reactivity was observed with the FdUMP precursor 5FU and with the natural nucleoside uridine and the nucleotides dTMP, dTTP and UTP. A considerable cross-reactivity was observed with the monophosphate of bromodeoxyuridine (Br-dUMP). Radio-immuno assays for FdUMP and dUMP were developed for which a 100-fold dilution could be used. The FdUMP assay was linear in a range of 0.1 to 5 pmol FdUMP in aqueous solutions. Tumour samples contained a non-identified interfering factor; a similar interference was observed in an enzyme based assay for FdUMP. The dUMP assay was performed by competition of unlabeled dUMP with tritiated FdUMP and was linear from 50 to 2000 pmol dUMP per assay. The antibody recognized FdUMP bound in a ternary complex synthesized in a cell-free system between FdUMP its target enzyme thymidylate synthase and the folate co-factor. Immunohistochemical staining for demonstration of the ternary complex in 5FU treated cells and tumours from patients and animals was not yet successful neither with peroxidase nor with immunofluorescence staining. Possibly the amount of bound FdUMP is below the detection limit or FdUMP bound to TS is masked. The antibody may prove to be useful in studies on modulation of FdUMP and dUMP after treatment with thymidylate synthase inhibitors.

Original languageEnglish (US)
Pages (from-to)835-839
Number of pages5
JournalAnticancer Research
Volume13
Issue number4
StatePublished - 1993
Externally publishedYes

Fingerprint

Fluorodeoxyuridylate
Fluorouracil
Antibodies
Thymidylate Synthase
Immune Sera
Uracil Nucleotides
Staining and Labeling
Uridine Triphosphate
Cell-Free System
2'-deoxyuridylic acid
Thyroglobulin

Keywords

  • 2'deoxyuridine-5'monophosphate
  • 5-fluoro-2'deoxyuridine-5'monophosphate
  • 5-fluorouracil
  • Thymidylate synthase

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Peters, G. J., Laurensse, E., Steinbusch, H. W. M., De Vente, J., Smid, K., Van der Wilt, C. L., & Pinedo, H. M. (1993). Development, characterization and application of an antibody against 5-fluoro-2'deoxyuridine-5'monophosphate, the active metabolite of 5-fluorouracil. Anticancer Research, 13(4), 835-839.

Development, characterization and application of an antibody against 5-fluoro-2'deoxyuridine-5'monophosphate, the active metabolite of 5-fluorouracil. / Peters, G. J.; Laurensse, E.; Steinbusch, H. W M; De Vente, J.; Smid, K.; Van der Wilt, C. L.; Pinedo, H. M.

In: Anticancer Research, Vol. 13, No. 4, 1993, p. 835-839.

Research output: Contribution to journalArticle

Peters, GJ, Laurensse, E, Steinbusch, HWM, De Vente, J, Smid, K, Van der Wilt, CL & Pinedo, HM 1993, 'Development, characterization and application of an antibody against 5-fluoro-2'deoxyuridine-5'monophosphate, the active metabolite of 5-fluorouracil', Anticancer Research, vol. 13, no. 4, pp. 835-839.
Peters, G. J. ; Laurensse, E. ; Steinbusch, H. W M ; De Vente, J. ; Smid, K. ; Van der Wilt, C. L. ; Pinedo, H. M. / Development, characterization and application of an antibody against 5-fluoro-2'deoxyuridine-5'monophosphate, the active metabolite of 5-fluorouracil. In: Anticancer Research. 1993 ; Vol. 13, No. 4. pp. 835-839.
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abstract = "5-Fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is the active metabolite of the anticancer agent 5-fluorouracil (5FU). Antibodies against a conjugate of thyroglobulin with acetylated FdUMP were raised in 4 rabbits. The maximal titer was reached after 3-5 boosters. Subsequently the antisera were collected and partially purified. In a competition assay a 100-fold excess of the natural nucleotide dUMP could displace tritiated FdUMP (20 pmol per assay) by about 50{\%} in all antisera. However tritiated dUMP itself did not bind to the antibody. No cross-reactivity was observed with the FdUMP precursor 5FU and with the natural nucleoside uridine and the nucleotides dTMP, dTTP and UTP. A considerable cross-reactivity was observed with the monophosphate of bromodeoxyuridine (Br-dUMP). Radio-immuno assays for FdUMP and dUMP were developed for which a 100-fold dilution could be used. The FdUMP assay was linear in a range of 0.1 to 5 pmol FdUMP in aqueous solutions. Tumour samples contained a non-identified interfering factor; a similar interference was observed in an enzyme based assay for FdUMP. The dUMP assay was performed by competition of unlabeled dUMP with tritiated FdUMP and was linear from 50 to 2000 pmol dUMP per assay. The antibody recognized FdUMP bound in a ternary complex synthesized in a cell-free system between FdUMP its target enzyme thymidylate synthase and the folate co-factor. Immunohistochemical staining for demonstration of the ternary complex in 5FU treated cells and tumours from patients and animals was not yet successful neither with peroxidase nor with immunofluorescence staining. Possibly the amount of bound FdUMP is below the detection limit or FdUMP bound to TS is masked. The antibody may prove to be useful in studies on modulation of FdUMP and dUMP after treatment with thymidylate synthase inhibitors.",
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AU - Laurensse, E.

AU - Steinbusch, H. W M

AU - De Vente, J.

AU - Smid, K.

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AU - Pinedo, H. M.

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