人肿瘤转移抑制基因TMSG-1单克隆抗体的制备、鉴定及在肿瘤检测中的应用

目的　明确人肿瘤转移抑制基因TMSG- 1蛋白分子量大小和细胞定位,制备和鉴定TMSG- 1单克隆抗体,并探讨其在临床肿瘤标本检测的价值。方法　采用多肽固相合成法 (Fmoc法 )合成 15肽TMSG -1抗原片段,并与匙孔槭血蓝蛋白 (mcKLH)偶联,免疫BALB/C小鼠,采用细胞融合、ELISA法检测和快速有限稀释法筛选和制备单克隆抗体,并应用Western印迹和免疫组织化学ABC法染色进行抗体鉴定和肿瘤检测。结果　筛选出抗TMSG- 1抗体的单克隆杂交瘤细胞系C8,免疫球蛋白亚类测定为IgM。半抗原竞争抑制实验证实该抗体是特异的抗TMSG- 1抗体。Western印迹显示不转移癌细胞亚系 2B4和LH7有明显的相对分子质量为 45 .000蛋白条带,而高转移亚系 1E8和BE1则条带很弱。细胞免疫组织化学染色显示 2B4和LH7强阳性,为细胞膜和细胞质着色;而 1E8和BE1则为阴性。石蜡切片ABC法检测 52例乳腺癌和 41例结肠癌组织中TMSG- 1蛋白表达发现:未转移的癌组织多为中度阳性或强阳性,中度以上阳性率分别为 36% (乳腺癌 )和 52. 4% (结肠癌 );而转移的癌组织多为弱阳性或阴性, 中度以上阳性率分别为 7 .4% (乳腺癌 )和 35% (结肠癌 ),统计学分析表明TMSG 1在未转移癌组织中的表达显著高于转移性癌组织 (P<0 .05)。结论　成功制备了抗人肿瘤转移抑制基

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). Conclusion 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.