Immunological quantitation of thymidylate synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex with the monoclonal antibody TS 106

Abstract

Thymidylate synthase (TS) is responsible for the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. One of the principal mechanisms of action of 5-fluorouracil (5-FU) is the inhibition of TS by formation of a ternary covalent complex consisting of TS-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate. We have developed a Western immunoblot assay using the monoclonal antibody TS 106 to measure ternary complex and free TS in intact human carcinoma cells following exposure to either 5-FU alone or 5-FU plus leucovorin. Lysates from cells treated with either 5-FU or 5-FU/leucovorin were resolved in 15% polyacrylamide gel, transferred onto nitrocellulose and immunoblotted using TS 106 antibody. Detection of positive bands was by a chromogenic substrate strain. Immunoblotting detected free TS at 36 kDa and TS in ternary complex at 38.5 kDa which were quantitated by densitometric scanning. This assay was able to detect a ternary complex from intact cells treated with 5-FU or 5-FU/leucovorin up to 96 h after drug removal. The ratio of complex to free TS was up to 2-fold greater in 5-FU/leucovorin-treated cells compared to those treated with 5-FU alone. This assay may be applied to measuring the formation and stability of ternary complex and free TS in patient tissue samples.