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目的研制针对肝细胞癌标志物磷脂酰肌醇蛋白聚糖-3(GPC3)的高特异抗体。方法用Biosun软件预测和分析GPC3的抗原表位肽,人工合成选定的表位肽,与载体蛋白KLH偶联后免疫新西兰大白兔制备抗血清。以抗原亲和层析法制取高纯抗GPC3抗体,对肝癌细胞株HepG2和胎盘组织进行Western blot印迹及对肝癌组织切片进行免疫组化染色,并用夹心ELISA对肝癌患者的血清进行了检测。结果选定并合成两条抗原表位肽(命名为GPC3-P1和GPC3-P2),取得高效价抗血清(分别为1:204 800和1:409 600)。两种抗GPC3抗体纯度均达到95%以上,在Western blot印迹能特异性地检测到GPC3;在免疫组化染色中均与肿瘤组织GPC3特异性结合,在正常组织中没有染色;夹心ELISA可以检测到肝癌患者血清中的GPC3。结论该两种抗GPC3不同表位的高特异抗体可用于研制相关肿瘤检测试剂盒。
Objective To develop a highly specific antibody against hepatocellular carcinoma marker glypican-3 (GPC3). Methods Biosun software was used to predict and analyze the epitope peptide of GPC3. The selected epitope peptide was synthesized and conjugated with the carrier protein KLH to immunize New Zealand white rabbits to prepare antiserum. High-purity anti-GPC3 antibody was prepared by antigen affinity chromatography, Western blotting was performed on HepG2 and placenta tissues of hepatocellular carcinoma cells, immunohistochemical staining was performed on the sections of hepatocellular carcinoma, and serum of liver cancer patients was detected by sandwich ELISA. Results Two epitope peptides (named as GPC3-P1 and GPC3-P2) were selected and synthesized to obtain high titer antisera (1: 204 800 and 1: 409 600, respectively). The purity of the two anti-GPC3 antibodies reached more than 95%, GPC3 was detected by Western blot. The immunohistochemical staining showed GPC3 specific binding to tumor tissue without staining in normal tissues. Sandwich ELISA could detect GPC3 in the serum of liver cancer patients. Conclusion The two highly specific antibodies against different epitopes of GPC3 can be used to develop related tumor detection kits.