[Monoclonal antibodies against human tumor metastasis suppressor gene-1 TMSG-1: preparation, characterization and application].
OBJECTIVE
In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.


METHODS
A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.


RESULTS
One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).


CONCLUSIONS
C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.
Pei
Fei
F
Department of Pathology, Health Science Center, Peking University, Beijing 100083, China.
You
Jiang-feng
JF
Ning
Jun-yu
JY
Yang
Jing-ping
JP
Wang
Yu-ping
YP
Han
Zhi-hui
ZH
Wang
Jie-liang
JL
Cui
Xiang-lin
XL
Yang
Shao-min
SM
Zheng
Jie
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D051049:Sphingosine N-Acyltransferase; D025521:Tumor Suppressor Proteins / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D025521:Tumor Suppressor Proteins; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase