肝细胞癌标志物GPC3表位肽抗体制备及其特性测定  

Epitope prediction and antibody preparation of glypican-3(GPC3)

作　　者：吴萌[1] 闫静辉[1] 张小兵[1] 李春生[1] 陈英珠[1] 程华[1] 李亚璞[1] 邢玉敏[2] 

机构地区：[1]河北省生物研究所,石家庄050081 [2]石家庄市中医院

出　　处：《中华微生物学和免疫学杂志》2007年第7期669-674,共6页Chinese Journal of Microbiology and Immunology

摘　　要：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 prepare the pure rabbit antibodies specific to glypican - 3 （ GPC 3 ） . Methods Predicting GPC3 epitopes with the Biesun software, synthesizing the aim epitope peptides and coupling them with KLH. New Zealand while rabbits were immunized with peptide-KLH respectively to produce antisera and the anti- sera were purified by antigen affinity chromatography to obtain antibodies specific to GPC3. The pure anti-GPC3 antibodies were identified by Western blot, immunohistochemical analysis and sandwich-ELISA. Results Two aim epitope peptides（named GPC3-P1 and GPC3-P2） were determined for antibody production, both prepared rabbit antisera revealed high titers （ 1 ： 204 800 and 1 ： 409 600, respectively）. The purity of two affinity-purified anti- GPC3 antibodies is over 95%. Both antibodies could combined specific to GPC3 in the tests of Western blot, immunohistochemical analysis and sandwich-ELISA. Conclusion Both anti-GPC3 antibodies are different epitope antibodies and may be used for the preparation of the related tumor test kits.

关 键 词：磷脂酰肌醇蛋白聚糖-3 肿瘤标志物 表位肽预测 抗原亲和层析 多克隆抗体 

分 类 号：R735.7[医药卫生—肿瘤]