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分泌人源性单克隆抗体永生化淋巴细胞系的构建 |
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邬淑云 温淦升 徐方云 刘秋如 龚文华 余鹰 妇产科 易为民 刘丝荪
摘 要 目的：构建分泌人源性单克隆抗体(McAb)的永生化B淋巴细胞系。方法：用正丁醇法提取的人卵巢癌细胞系ao10/17抗原在含免疫活化剂的无血清培基中免疫人淋巴细胞；将制备ao 10/17细胞DNA转染经体外免疫的淋巴细胞。采用ELISA筛选和有限稀释法克隆化。结果：建成4株分泌抗卵巢癌McAb的转染体细胞AT1、AT2、AT3和AT4；McAb相加试验表明，它们识别卵巢癌抗原分子的同一表位。AT4已在体外培养460多天，冻存复苏后仍保持抗体产生能力。夹心ELISA发现，人源性McAb AT4能与卵巢癌细胞表面可溶性抗原发生免疫反应。结论：体外免疫和DNA转染的结合，可建成分泌人源性McAb的永生化B淋巴细胞系，为人源性McAb的产生提供了一条技术途径。
主题词 淋巴细胞；抗体，单克隆；转染；免疫法；卵巢肿瘤
The construction of immortalized lymphoid cell lines secreting
human monoclonal antibodies
WU Su-Yun, WEN Gan-Sheng, XU Fang-Yun, LIU Qiu-Ru,
GONG Wen-Hua, Yu Ying, YI Wei-Min△, LIU Si-Sun△
Department of Pathophysiology and Tumor Immunology Research, Jiangxi Medical College, Nanchang(330006)
△Department of Obstetrics and Gynecology First Affiliated Hospital, Jiangxi Medical College
Abstract AIM:To construct immortalized lymphoid cell lines secreting human monoclonal antibodies. METHODS：Human lymphocytes were immunized in serum-free media containing immunoactive reagents using ovarian cancer cell line ao10/17 antigen extracted by 1-butanol. In vitro immunized lymphocytes were transfected with DNA preparations from ao 10/17. RESULTS：Four transfectant cell lines designated AT1, AT2, AT3 and AT4 secreting monoclonal antibodies against ovarian cancer were established by ELISA-based screening and limiting dilution. All of monoclonal antibodies were reactive with the identical epitopes of ovarian cancer-associated antigen molecule in the addition test. The transfectant cell line AT4 has grown continuously in vitro culture for more than 460 days and maintained the ability of secreting antibody after frozen and recovered. Human monoclonal antibody AT4 had immunoreactions with a soluble antigen from the cell surface of ovarian cancer by using sandwich ELISA.CONCLUSION：A combination of in vitro immunization and transfection with DNA induced the construction of B-cell-derived cell lines w[1] [2] 下一页
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