DEVELOPMENT, CHARACTERIZATION AND APPLICATION OF AN ANTIBODY AGAINST 5-FLUORO-2'DEOXYURIDINE-5'MONOPHOSPHATE, THE ACTIVE METABOLITE OF 5-FLUOROURACIL

5-Fluoro-2' -deoxyuridine-5' -monophosphate (FdUMP) is the active meta bolite of the anticancer agent 5-fluorouracil (5FU). Antibodies agains t a conjugate of thyroglobulin with acetylated FdUMP were raised in 4 rabbits. The maximal titer was reached after 3-5 boosters. Subsequentl y, the antisera were collected and partially purified. In a competitio n assay a 100-fold excess of the natural nucleotide dUMP could displac e tritiated FdUMP (20 pmol per assay) by about 50% in all antisera. Ho wever, tritiated dUMP itself did not bind to the antibody. No cross-re activity was observed with the FdUMP precursor 5FU and with the natura l nucleoside uridine and the nucleotides dTMP, dTTP and UTP. A conside rable cross-reactivity was observed with the monophosphate of bromodeo xyuridine (Br-dUMP). Radio-immuno assays for FdUMP and dUMP were devel oped, 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. Tumou r samples contained a non-identified interfering factor; a similar int erference was observed in an enzyme based assay for FdUMP. The dUMP as say was performed by competition of unlabeled dUMP with tritiated FdUM P and was linear from 50 to 2000 pmol dUMP per assay. The antibody rec ognized FdUMP bound in a ternary complex synthesized in a cell-free sy stem between FdUMP, its target enzyme thymidylate synthase and the fol ate co-factor Immunohistochemical staining for demonstration of the te rnary complex in 5FU treated cells and tumours from patients and anima ls was not yet successful, neither with peroxidase nor with immunofluo rescence staining. Possibly the amount of bound FdUMP is below the det ection 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.