Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity1

Testis-specific protease 50 (TSP50) is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSP50 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector pcDNA3. 1. Rabbit anti-TSP50 polyclonal antibodies were prepared by means of intramuscular injection of pcDNA3. 1-TSP50 into the rabbits. Titers of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSP50 fusion protein as an antigen. In addition, we examined the expression of TSP50 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.