Immunoassay for thymidylate synthase in cytosols from human colorectal-carcinoma cell-lines

Thymidylate synthase (TS) is an essential enzyme for DNA biosynthesis that is an important target of fluoropyrimidine anti-cancer drugs such as 5-fluorouracil (5-FU), which is frequently used against human colon adenocarcinomas. Because levels of TS in such tumors may be predictive of chemotherapeutic response to 5-FU, it is desirable to have a method for TS quantitation simpler than the laborious conventional biochemical assays that currently exist. We therefore describe a rapid and simple immunoassay for quantitating human TS (in cell or tissue extracts) that employs a rabbit antibody to TS from bacteriophage T4 and an automated colorimetric readout. The sensitivity of the assay is demonstrated by its ability to show a different TS level in cytosols from each of 8 human colorectal carcinoma cell lines tested and a 2.5-fold difference between the highest and lowest values observed. Western immunoblot was used to show that the rabbit antibody to T4 TS cross-reacts very specifically with human TS enzyme. This indicates that some evolutionarily conserved (phage or bacterial) enzymes, not purified from human sources, may be useful as antigens in producing anti-sera for human diagnostic tests.