【摘要】 建立杂交瘤单抗亲和层析纯化抗原、体外抗原致敏淋巴细胞和RT PCR克隆人抗体基因的技术 .将亲和层析纯化的大肠癌相关抗原CA Hb3经SDS PAGE和免疫印迹鉴定后 ,与IL 2和丝裂原于体外致敏大肠癌病人 1 0ml外周血淋巴细胞 (PBL) ,出现淋巴母细胞化和集落形成现象 .致敏PBL的总RNA比未致敏PBL的量增加了 2 5倍 .致敏后RT PCR扩增的人抗体VH CH1 (IgG)和VL CL(κ)基因的量比未致敏者多 1 3倍 .该技术可将鼠源杂交瘤单抗人源化 .更多还原

【Abstract】 A strategy for cloning human immunoglobulin genes with antigen in vitro immunization and RT-PCR was established. Colorectal carcinoma-associated antigen CA-Hb3 were purified through a monoclonal antibody Hb3-Sepharose 4B affinity column and analyzed by SDS-PAGE and Western blot. Peripheral blood lymphocytes (PBL) from 10 ml blood of a patient with colorectal carcinoma were immunized with CA-Hb3, rhIL-2 and pokeweed mitogen. The lymphoblast-like cells and colonies could been seen after immunization. The amounts of total RNA from the immunized PBL are 25 times more than that from the non- immunized. Human immunoglobulin, VH-CH1 (IgG) and VL-CL (κ), genes were amplified with RT-PCR with the above total RNA or mRNA as templates. The products of human immunoglobulin genes from the immunized PBL are 13 times more than that from the non-immunized. This strategy would be used for humanizing mouse-original monoclonal antibodies.更多还原