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

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【摘要】：背景：流行病學(xué)調(diào)查顯示，心力衰竭在成人住院病因中排名第四，嚴(yán)重危害著人類健康。自腦鈉肽(brain natriuretic peptide，BNP)被發(fā)現(xiàn)以來(lái)至今，BNP在心力衰竭、急性冠脈綜合征等疾病中的診斷價(jià)值、判斷預(yù)后價(jià)值等已經(jīng)得到了廣大學(xué)者的認(rèn)可，美國(guó)心臟病學(xué)會(huì)、歐洲心臟病學(xué)會(huì)也分別將BNP檢測(cè)列入其心力衰竭診療指南，BNP有望成為心力衰竭診斷、危險(xiǎn)分層、預(yù)后評(píng)判的標(biāo)記物。 目的：建立分泌BNP單克隆抗體雜交瘤細(xì)胞株，制備抗人BNP單克隆抗體，為建立BNP檢測(cè)方法及其臨床應(yīng)用提供基礎(chǔ)。 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(qiáng)免疫小鼠，共加強(qiáng)免疫3次，每次間隔3周。免疫完成后2周對(duì)小鼠采血，間接酶聯(lián)免疫吸附實(shí)驗(yàn)(enzyme-linked immunosorbent assay，ELISA)法檢測(cè)小鼠血清BNP抗體滴度。至融合前3天以無(wú)佐劑的100μg完全抗原再次加強(qiáng)免疫。 約1×10~8個(gè)免疫小鼠脾細(xì)胞在聚乙二醇作用下，與2-5×10~7骨髓瘤細(xì)胞SP2／0融合。融合后細(xì)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(cè)細(xì)胞培養(yǎng)上清BNP抗體，篩選出BNP抗體陽(yáng)性的雜交瘤細(xì)胞以有限稀釋法亞克隆培養(yǎng)。連續(xù)進(jìn)行三次亞克隆，篩選出最終呈單克隆生長(zhǎng)、穩(wěn)定分泌BNP抗體的細(xì)胞株，選取一株行染色體核型分析。 擴(kuò)大培養(yǎng)分泌BNP單克隆抗體的細(xì)胞株，以5×10~5-1×10~6個(gè)細(xì)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(kāi)
[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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 顧東風(fēng) ,黃廣勇 ,吳錫桂 ,段秀芳 ,何江 ,Paul K Whelton ,Stephen Mac Mahon;中國(guó)心力衰竭流行病學(xué)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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