Characterization of a polyclonal antibody to human thymidylate synthase suitable for the study of colorectal cancer specimens

Measurement of thymidylate synthase (hTS) using immunohistochemical techniq ues has been reported in several clinical studies. However, its Value as a prognostic indicator is still not clear. To pursue this, we have developed a new rabbit polyclonal antibody, hTS7.4. The antigen was recombinant hTS c ontaining an N-terminal His(6)-tag. Antiserum hTS7.4 detected recombinant h TS by ELISA at a titer of 1:100,000. Western blot analysis of several human cell lines showed a single band of the expected 36-kD molecular size. HeLa cells treated with the TS inhibitor 5-FUdR showed the expected additional band corresponding to the ternary complex of hTS-dFUMP-reduced folate. hTS7 .4 detected TS in bacterial, rat, mouse, and monkey cell extracts, and hTS8 .3 (a closely related antiserum) immunoprecipitated a 36-kD [S-35]-methioni ne-labeled protein from HeLa extracts. TS was detectable by indirect immuno fluorescence in HeLa cells. Proliferating normal human fibroblasts in cultu re showed staining, but nonproliferating cells did not. Lymphocytes in the germinal center of human tonsil tissue, which are known to be proliferating , stained with hTS7.4 and also with monoclonal antibody TS106. TS may there fore be useful as an immunohistochemical marker of cell proliferation. Norm al colon mucosa showed weak staining, whereas some colorectal cancer specim ens stained very strongly with hTS7.4. A clinical study of colorectal cance r using this antibody is in progress.