抗大肠癌噬菌体单链抗体的筛选及初步鉴定

 应用 3种方法 (肿瘤细胞膜表面和胞内、裸鼠体内和组织切片 ) ,从全人源化的抗大肠癌噬菌体初级抗体库中筛选肿瘤特异性的噬菌体单链抗体 (Sc Fv) .在肿瘤细胞经过 3轮亲和选择 ,回收结合胞膜和内化进入胞内的噬菌体 ,得到抗肿瘤噬菌体单链抗体的富集倍数为 430倍 ;荷瘤裸鼠体内注入初级抗体库后 ,在不同时刻点处死裸鼠 ,回收肿瘤组织内的噬菌体 ,其回收率在 2 4 h时最高 ;初级抗体库与大肠癌组织切片亲和选择后 ,从冰冻组织切片上比从石蜡组织切片上回收得到的噬菌体高出约 1 .6倍 .从上述方法挑选单克隆 ,经 ELISA筛选抗大肠癌阳性噬菌体克隆株 ,分离得到 5个对大肠癌细胞反应较好的单克隆噬菌体单链抗体 .进一步用细胞 ELISA检测对各种肿瘤细胞的特异性反应 ,其中 4个对大肠癌细胞有很好的特异性 ,1个克隆对所有肿瘤细胞均有反应 .因此 ,3种方法用于筛选抗大肠癌噬菌体初级抗体库是有效的 ,具有推广和应用价值 .

Selection of Single-chain Fv Antibodies to Colorectal Cancer Antigens and Preliminary Characterization

To isolate tumor specific phage antibodies from single chain Fv fusion phage library that was constructed from B lymphocytes of patients with colorectal cancer,selections of phage antibody library were performed on complex antigen sources such as whole live cells or tissue sections,or even by in vivo selection methods.First,the library was subjected to three rounds of positive selection on human colorectal cancer cells and two rounds of negative selection on human fibroblast cells and peripheral blood mononuclear cells (PBMC).Phages that bound cell membrane and that were internalized into cells were recovered.Enrichment of membrane bound and internalized phages was 430 times.Second, in vivo selections were performed in LS174T/HRT 18 xenografted mice.After phage library was injected into nude mice,specific phages were retrieved from tumor tissues at the variable time point of the experiments.The efficiency of phage recovery was the best at the time point of 24 hours.Finally,for selection on tissue sections,phages recovered on tissue cryosections were 1.6 times higher than those on tissue paraffin sections.Clones of phage antibodies were tested by cell ELISA,and 5 anti colorectal cancer phage antibody clones were isolated.Clones of 4 of 5 ScFv phages demonstrated specific tumor reactivity to 4 different colorectal cancer cell lines but not to 7 other tumor cell lines such as lung,liver,ovarian,nasopharyngeal and cervical cancer,and one reacted to all cancer cell lines,specifically.Three selection approaches used were suitable for selection of anti tumor phage antibodies.