本文關鍵詞：抗人腦鈉肽單克隆抗體的研制　出處：《南京醫(yī)科大學》2006年碩士論文　論文類型：學位論文

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【摘要】：背景：流行病學調(diào)查顯示，心力衰竭在成人住院病因中排名第四，嚴重危害著人類健康。自腦鈉肽(brain natriuretic peptide，BNP)被發(fā)現(xiàn)以來至今，BNP在心力衰竭、急性冠脈綜合征等疾病中的診斷價值、判斷預后價值等已經(jīng)得到了廣大學者的認可，美國心臟病學會、歐洲心臟病學會也分別將BNP檢測列入其心力衰竭診療指南，BNP有望成為心力衰竭診斷、危險分層、預后評判的標記物。 目的：建立分泌BNP單克隆抗體雜交瘤細胞株，制備抗人BNP單克隆抗體，為建立BNP檢測方法及其臨床應用提供基礎。 方法：合成的BNP與牛血清白蛋白(bovine serum albumin，BSA)以碳化二乙胺(EDC)法偶聯(lián)，制備BNP-BSA完全抗原。將BNP-BSA(100μgBNP)與等體積福氏完全佐劑混合后，腹腔注射Balb／c小鼠初次免疫，，3周后以BNP-BSA與等體積福氏不完全佐劑混合繼續(xù)加強免疫小鼠，共加強免疫3次，每次間隔3周。免疫完成后2周對小鼠采血，間接酶聯(lián)免疫吸附實驗(enzyme-linked immunosorbent assay，ELISA)法檢測小鼠血清BNP抗體滴度。至融合前3天以無佐劑的100μg完全抗原再次加強免疫。 約1×10~8個免疫小鼠脾細胞在聚乙二醇作用下，與2-5×10~7骨髓瘤細胞SP2／0融合。融合后細胞先后培養(yǎng)于次黃嘌呤、氨基喋呤、胸腺嘧啶核苷(hypoxanthine、aminopterin、thymidine，HAT)以及次黃嘌呤、胸腺嘧啶核苷(HT)選擇性培養(yǎng)液。培養(yǎng)10天后間接ELISA法檢測細胞培養(yǎng)上清BNP抗體，篩選出BNP抗體陽性的雜交瘤細胞以有限稀釋法亞克隆培養(yǎng)。連續(xù)進行三次亞克隆，篩選出最終呈單克隆生長、穩(wěn)定分泌BNP抗體的細胞株，選取一株行染色體核型分析。 擴大培養(yǎng)分泌BNP單克隆抗體的細胞株，以5×10~5-1×10~6個細胞／只接種于一周前腹腔注射0.5ml／只液體石蠟油的Balb／c小鼠。接種后7天開
[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 .

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

【參考文獻】

相關期刊論文 前1條

1 顧東風 ,黃廣勇 ,吳錫桂 ,段秀芳 ,何江 ,Paul K Whelton ,Stephen Mac Mahon;中國心力衰竭流行病學調(diào)查及其患病率[J];中華心血管病雜志;2003年01期

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