人绒毛膜促性腺激素单克隆抗体的制备与分离纯化

发布时间：2018-04-24 00:09

本文选题：人绒毛膜促性腺激素 + 单克隆抗体　；参考：《浙江工业大学》2010年硕士论文

【摘要】：人绒毛膜促性腺激素(human chorionic gonadotrophin, hCG)是人类受孕后胎盘合体滋养层细胞分泌的一种糖蛋白激素。在妊娠早期hCG的浓度迅速增高,检测尿中的hCG即可证实妊娠。有许多研究表明,在肿瘤病人的血和尿中有游离的hCG的β亚基存在。因此,研制高纯度的hCG单克隆抗体对早孕、癌症的检测有重要的意义。本研究利用经典的淋巴细胞融合技术,制备了人绒毛膜促性腺激素单克隆抗体,并采用不同方法进行纯化,通过比较和优化,建立了良好的纯化hCG单克隆抗体方法,为临床的快速诊断奠定基础,并为其他单克隆抗体的纯化提供了一定的参考价值。主要实验结果如下: 1.得到两株能稳定分泌hCG单克隆抗体的杂交瘤细胞株,细胞培养上清液中抗体效价达10 3以上,腹水抗体效价达10 7以上。 2.弗氏不完全佐剂预处理的小鼠比石蜡预处理的小鼠腹水产量高;10～12周龄小鼠腹水产量高于6～8周龄小鼠;雄性小鼠腹水产量高于雌性小鼠。 3.腹水经辛酸—硫酸铵沉淀法纯化后,抗体纯度大于80%,抗体回收率达40%,纯化后抗体的生物学活性没有下降。 4.腹水以磷酸缓冲液(pH7.0,20 mmol/L PB + 3 mol/L NaCl)上样于HiTrap rProtein A FF预装亲和层析柱,pH3.0,0.1 mol/L柠檬酸缓冲液一步洗脱,回收抗体的纯度大于98%,抗体回收率达75%,纯化后抗体的生物学活性没有下降。
[Abstract]:Human chorionic gonadotrophin (hCGG) is a glycoprotein hormone secreted by human placental syncytiotrophoblast cells. The concentration of hCG increases rapidly in early pregnancy. Detection of hCG in urine can confirm pregnancy. Many studies have shown the presence of free 尾-subunits of hCG in the blood and urine of cancer patients. Therefore, the development of high purity hCG monoclonal antibody is of great significance for the detection of early pregnancy and cancer. In this study, monoclonal antibodies against human chorionic gonadotropin were prepared by classical lymphocyte fusion technique and purified by different methods. A good method for purification of hCG monoclonal antibody was established by comparison and optimization. It provides a basis for rapid clinical diagnosis and provides some reference value for the purification of other monoclonal antibodies. The main results are as follows: 1. Two hybridoma cell lines which can secrete hCG monoclonal antibody stably were obtained. The titer of antibody in the supernatant of cell culture was over 103 and the antibody titer in ascites was over 107. 2. The ascites production of mice pretreated with Freund's incomplete adjuvant was higher than that of mice pretreated with paraffin wax. The ascites production of mice aged 10 ~ 12 weeks was higher than that of mice of 6 ~ 8 weeks old, and that of male mice was higher than that of female mice. 3. After the ascites were purified by octanoic acid-ammonium sulfate precipitation, the purity of the antibody was more than 80 and the recovery rate of the antibody was 40. The biological activity of the antibody was not decreased after purification. 4. Ascites were eluted on the HiTrap rProtein AFF pre-packed affinity chromatography column with pH 3.0 ~ (0.1) mol/L citric acid buffer at pH 7.0 ~ 20 mmol/L PB3 mol/L NaCl1. The purity of recovered antibody was greater than 98. The recovery rate of antibody was 75%, and the biological activity of purified antibody was not decreased.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】