Monoclonal antibodies against human tumor metastasis suppressor gene-1 (TMSG-1): Preparation, characterization, and application

To better understand the molecular mechanism of metastasis, the monoclonal antibody against tumor metastasis suppressor gene - 1 (TMSG-1, one novel gene) was prepared, characterized, and applied in estimating the metastatic potential of human tumors. A dominant epitope, TMSG-1(15) - derived from TMSG-1 - was automatically synthesized based on the Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen solution was used to immunize BALB/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and their characterization was performed by Western blotting and immunohistochemical staining. The hybridoma cell line secreting anti - TMSG-1 antibody, designated C8, was established after primary ELISA screening and consequent rapid limited dilution. C8 was IgM in isotyping. The competitive inhibition assay showed that the antibody was TMSG-1 specific. Furthermore, in Western blotting, a protein band of about 45 kD was detected with non-metastatic variant PC-3M-2B4 and PG-LH7 cells, but not with the isogenetic metastatic variant PC-3M-1E8 and PG-BE1 cells. Immunohistochemistry method showed that positive staining presented in the cytomembrane and cytoplasm of 2B4 and LH7 cells, while 1E8 and BE1 cells were non-reactive. The sections from paraffin-embedded blocks of human cancer ( 52 cases of breast carcinoma and 41 cases of colon cancer) were stained, showing strongly positive in non-metastatic tumor and weakly positive or negative in metastatic tumor; the strongly positive rate of TMSG-1 expression in human cancers were, respectively, 36%, 7.4%, 52.4%, and 35% in non-metastatic and metastatic breast cancer and non-metastatic and metastatic colon cancer ( p = 0.02). The monoclonal antibody developed against synthetic peptide was TMSG-1 specific, and it has promise in Western blot and immunohistochemistry for detecting the TMSG-1 expression of cancer cells and tissues. It may provide an important tool in the study of TMSG-1 expression and function in both experimental and clinical studies. Furthermore, our tests confirmed that TMSG-1 protein has excellent inverse correlation to tumor metastasis potential, with the molecular weight of 45 kD ( supporting the encoded protein containing 380 amino acids) and localization in cytomembrane and cytoplasm of tumor cell.