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目的通过鉴定制备的人肝素酶单克隆抗体,筛选出具有抗肿瘤转移特异性的单克隆抗体株,并大量制备。方法采用Western blot技术,以HepG2、SGC-7901、MKN45、SW480、U2OS、MCF-7细胞总蛋白为样本,对已制备的3株人肝素酶单克隆抗体进行初步鉴定,筛选出能够与肝素酶蛋白特异性结合的抗体,并采用免疫细胞化学技术进一步验证筛选出的抗体特异性,然后通过Transwell体外侵袭实验观察筛选出的肝素酶单克隆抗体对肿瘤转移的抑制作用。结果已制备的3株人肝素酶单克隆抗体均能与不同肿瘤细胞肝素酶蛋白结合,其中12号抗体与商品化肝素酶单克隆抗体在MCF-7细胞中均检测出肝素酶弱阳性表达,其特异性最佳;免疫细胞化学结果也显示在SGC-7901、HepG2、MKN45、SW480和U2OS细胞中12号抗体能检测出肝素酶阳性表达,而在MCF-7细胞中检测出肝素酶弱阳性表达;Transwell体外侵袭实验结果显示12号抗体(100μg,浓度约1 mg/ml)作用于SGC-7901、HepG2、MKN45、SW480和U2OS细胞48 h后,其穿膜细胞数与正常小鼠IgG对照组相比明显减少(P<0.05)。结论纯化制备的人肝素酶单克隆抗体能够与肿瘤细胞表达的肝素酶蛋白特异性结合,并通过抑制肝素酶蛋白达到抑制肿瘤细胞的扩散与转移。
OBJECTIVE To identify monoclonal antibody against human heparanase and to screen monoclonal antibody against tumor metastasis and to prepare it in large quantities. Methods The total proteins of HepG2, SGC-7901, MKN45, SW480, U2OS and MCF-7 cells were detected by Western blot. The monoclonal antibodies against human heparanase were preliminarily identified, Enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry was used to further validate the specificity of the selected antibodies. The inhibition of tumor metastasis by monoclonal antibody against Heparanase was observed by Transwell in vitro invasion assay. Results The three monoclonal antibodies against human heparanase were able to bind to different hepatic cell heparanase proteins. Among them, antibody No. 12 and commercial heparanase monoclonal antibody detected heparanase in MCF-7 cells The results of immunocytochemistry also showed that Heparinase-positive expression was detected in SGC-7901, HepG2, MKN45, SW480 and U2OS cells, while Heparinase-positive was detected in MCF-7 cells Heparinase was weakly positive. Transwell invasion assay showed that the number of transmembrane cells in SGC-7901, HepG2, MKN45, SW480 and U2OS cells treated with antibody 12 (100μg, concentration 1 mg / ml) Compared with the normal mouse IgG control group significantly reduced (P <0.05). Conclusion The purified monoclonal antibody of human heparanase can specifically bind heparinase protein expressed in tumor cells and inhibit the proliferation and metastasis of tumor cells by inhibiting heparinase protein.