本文選題：人絨毛膜促性腺激素 + 單克隆抗體��； 參考：《浙江工業(yè)大學(xué)》2010年碩士論文

【摘要】：人絨毛膜促性腺激素(human chorionic gonadotrophin, hCG)是人類受孕后胎盤合體滋養(yǎng)層細胞分泌的一種糖蛋白激素。在妊娠早期hCG的濃度迅速增高,檢測尿中的hCG即可證實妊娠。有許多研究表明,在腫瘤病人的血和尿中有游離的hCG的β亞基存在。因此,研制高純度的hCG單克隆抗體對早孕、癌癥的檢測有重要的意義。本研究利用經(jīng)典的淋巴細胞融合技術(shù),制備了人絨毛膜促性腺激素單克隆抗體,并采用不同方法進行純化,通過比較和優(yōu)化,建立了良好的純化hCG單克隆抗體方法,為臨床的快速診斷奠定基礎(chǔ),并為其他單克隆抗體的純化提供了一定的參考價值。主要實驗結(jié)果如下: 1.得到兩株能穩(wěn)定分泌hCG單克隆抗體的雜交瘤細胞株,細胞培養(yǎng)上清液中抗體效價達10 3以上,腹水抗體效價達10 7以上。 2.弗氏不完全佐劑預(yù)處理的小鼠比石蠟預(yù)處理的小鼠腹水產(chǎn)量高;10～12周齡小鼠腹水產(chǎn)量高于6～8周齡小鼠;雄性小鼠腹水產(chǎn)量高于雌性小鼠。 3.腹水經(jīng)辛酸—硫酸銨沉淀法純化后,抗體純度大于80%,抗體回收率達40%,純化后抗體的生物學(xué)活性沒有下降。 4.腹水以磷酸緩沖液(pH7.0,20 mmol/L PB + 3 mol/L NaCl)上樣于HiTrap rProtein A FF預(yù)裝親和層析柱,pH3.0,0.1 mol/L檸檬酸緩沖液一步洗脫,回收抗體的純度大于98%,抗體回收率達75%,純化后抗體的生物學(xué)活性沒有下降。
[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.
【學(xué)位授予單位】：浙江工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：1794245