抗人脑钠肽单克隆抗体的研制

发布时间：2018-01-08 16:20

本文关键词：抗人脑钠肽单克隆抗体的研制　出处：《南京医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：背景：流行病学调查显示，心力衰竭在成人住院病因中排名第四，严重危害着人类健康。自脑钠肽(brain natriuretic peptide，BNP)被发现以来至今，BNP在心力衰竭、急性冠脉综合征等疾病中的诊断价值、判断预后价值等已经得到了广大学者的认可，美国心脏病学会、欧洲心脏病学会也分别将BNP检测列入其心力衰竭诊疗指南，BNP有望成为心力衰竭诊断、危险分层、预后评判的标记物。目的：建立分泌BNP单克隆抗体杂交瘤细胞株，制备抗人BNP单克隆抗体，为建立BNP检测方法及其临床应用提供基础。方法：合成的BNP与牛血清白蛋白(bovine serum albumin，BSA)以碳化二乙胺(EDC)法偶联，制备BNP-BSA完全抗原。将BNP-BSA(100μgBNP)与等体积福氏完全佐剂混合后，腹腔注射Balb／c小鼠初次免疫，，3周后以BNP-BSA与等体积福氏不完全佐剂混合继续加强免疫小鼠，共加强免疫3次，每次间隔3周。免疫完成后2周对小鼠采血，间接酶联免疫吸附实验(enzyme-linked immunosorbent assay，ELISA)法检测小鼠血清BNP抗体滴度。至融合前3天以无佐剂的100μg完全抗原再次加强免疫。约1×10~8个免疫小鼠脾细胞在聚乙二醇作用下，与2-5×10~7骨髓瘤细胞SP2／0融合。融合后细胞先后培养于次黄嘌呤、氨基喋呤、胸腺嘧啶核苷(hypoxanthine、aminopterin、thymidine，HAT)以及次黄嘌呤、胸腺嘧啶核苷(HT)选择性培养液。培养10天后间接ELISA法检测细胞培养上清BNP抗体，筛选出BNP抗体阳性的杂交瘤细胞以有限稀释法亚克隆培养。连续进行三次亚克隆，筛选出最终呈单克隆生长、稳定分泌BNP抗体的细胞株，选取一株行染色体核型分析。扩大培养分泌BNP单克隆抗体的细胞株，以5×10~5-1×10~6个细胞／只接种于一周前腹腔注射0.5ml／只液体石蜡油的Balb／c小鼠。接种后7天开
[Abstract]:Background : The epidemiological investigation showed that heart failure ranked fourth in the etiology of adult hospitalization and seriously jeopardized the human health . Since brain natriuretic peptide ( BNP ) has been found , BNP has been recognized by scholars in heart failure , acute coronary syndrome and so on . BNP has been recognized by the American Society of Cardiology and the European Society of Cardiology . BNP is expected to be a marker of heart failure diagnosis , risk stratification and prognosis evaluation . Objective : To establish a hybridoma cell line secreting BNP monoclonal antibody and to prepare monoclonal antibody against human BNP . Methods : The BNP and bovine serum albumin ( BSA ) were coupled with bovine serum albumin ( BSA ) to prepare the complete antigen of BNP - BSA . The spleen cells of approximately 1 脳 10 ~ 8 immune mice were fused with SP2 / 0 cells of 2 - 5 脳 10 ~ 7 myeloma cells under the action of polyethylene glycol . After fusion , the cells were cultured in hypoxanthine , aminopterin , thymidine ( hypoxanthine , aminopterin , thymidine , hat ) and hypoxanthine , thymidine ( HT ) selective culture . After 10 days of culture , the cells were cultured in a limited dilution method . The cell strain secreting BNP monoclonal antibody was expanded to 5 脳 10 ~ 5 - 1 脳 10 ~ 6 cells / 2 Balb / c mice injected with 0.5 ml / L paraffin oil only once a week . 7 days after inoculation .

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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