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Journal of Histochemistry and Cytochemistry, Vol. 47, 1563-1574, December 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Characterization of a Polyclonal Antibody to Human Thymidylate Synthase Suitable for the Study of Colorectal Cancer Specimens

Arsalan S. Haqqania, Randy T. Cowlinga, Jean A. Marounb, and H. Chaim Birnboima,b
a Department of Biochemistry, Microbiology and Immunology, Ottawa, Ontario, Canada
b University of Ottawa, and the Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada

Correspondence to: H. Chaim Birnboim, Ottawa Regional Cancer Centre, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6

Measurement of thymidylate synthase (hTS) using immunohistochemical techniques 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 containing an N-terminal His6-tag. Antiserum hTS7.4 detected recombinant hTS 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 [35S]-methionine-labeled protein from HeLa extracts. TS was detectable by indirect immunofluorescence in HeLa cells. Proliferating normal human fibroblasts in culture 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 therefore be useful as an immunohistochemical marker of cell proliferation. Normal colon mucosa showed weak staining, whereas some colorectal cancer specimens stained very strongly with hTS7.4. A clinical study of colorectal cancer using this antibody is in progress. (J Histochem Cytochem 47:1563–1573, 1999)

Key Words: thymidylate synthase, polyclonal antibody, Western blot, immunohistochemistry, human, colorectal cancer, cell proliferation marker


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