利用改良后的脾内免疫和半固体培养基法制备单克隆抗体

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 166-169.

利用改良后的脾内免疫和半固体培养基法制备单克隆抗体

任瑞敏¹, 王云龙^{1, 2}, 张怡青⁴, 李恒思³, 王国强³, 李玉林³, 王继创³

（1.郑州大学生物工程系，郑州 450001；2.郑州职业技术学院，郑州 450121；3.河南省生物工程技术研究中心，郑州 450001；4.河南师范大学生命科学学院，新乡 453003）

收稿日期:2013-02-19 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:任瑞敏，女，硕士，研究方向：生物制药；E-mail：biorrm@126.com

The Improved Intrasplenic Immunization and Semi-solid Medium to Prepare Monoclonal Antibody

Ren Ruimin^{1 Wang Yunlong^{1, 2}, Zhang Yiqing⁴, Li Hengsi³, Wang Guoqiang³, Li Yulin³, Wang Jichuang³}

（1. Biological Engineering Department，Zhengzhou University，Zhengzhou 450001；2. Zhengzhou Technical College，Zhengzhou 450121；3. Henan Biotechnology Research Center，Zhengzhou 450001；4.^{College of Life Science, Henan Normal University, Xinxiang 453003）}

Received:2013-02-19 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 旨在利用改良后的脾内免疫和半固体培养基法制备单克隆抗体，节约免疫原用量、简化试验操作、一步法筛选和克隆杂交瘤细胞、缩短单抗制备周期。通过局部麻醉小鼠脾脏区域皮肤，切开后透过腹膜直接向脾脏内注射微量抗原，使小鼠的脾细胞在短时间内产生免疫反应，尾静脉注射强化免疫后进行细胞融合；选取半固体培养基（甲基纤维素、软琼脂）法，以有限稀释法作对照筛选分泌高效价抗体的杂交瘤细胞。结果显示，与传统的单抗制备方法相比，改进后的脾内免疫操作更简单，联合尾静脉注射，使用微量免疫原在短时间内产生高效价抗体；半固体培养基甲基纤维素浓度在1.1%条件下，获得克隆团较多，阳性率较高。本研究方法适用于大规模制备单克隆抗体。

关键词: 脾内免疫, 软琼脂培养法, 甲基纤维素法

Abstract: It was to utilize the improved intrasplenic immunization and semi-solid culture medium to reduce the immune dosage, clone the hybridoma simply, and shorten the period of cycle. The skin areas of mice spleen was anesthetized, then the spleen was injected with trace antigen, which could be immuned in a short period of time, cell fusion was prepared after intravenous injection. The three different methods（limited dilution, methylcellulose, soft agar）were used to get hybridomas which can secret monoclonal antibody. Results showed that compared with the traditional method of preparation of monoclonal antibody, the improved intrasplenic immunization was simpler operation, without the addition of adjuvant, and the high titer of antibody was getted in a short time. Positive rate was the highest when methylcellulose concentration in the 1.1% condition. It was suggested that these methods could be suitable for large scale preparation of monoclonal antibody.

Key words: Intrasplenic immunization, Soft agar, Methylcellulose

任瑞敏, 王云龙, 张怡青, 李恒思, 王国强, 李玉林, 王继创. 利用改良后的脾内免疫和半固体培养基法制备单克隆抗体[J]. 生物技术通报, 2013, 0(8): 166-169.

Ren Ruimin^{Wang Yunlong, Zhang Yiqing, Li Hengsi, Wang Guoqiang, Li Yulin, Wang Jichuang. The Improved Intrasplenic Immunization and Semi-solid Medium to Prepare Monoclonal Antibody[J]. Biotechnology Bulletin, 2013, 0(8): 166-169.}