本文選題：HoxA1 + 單克隆抗體　； 參考：《第四軍醫(yī)大學》2011年碩士論文

【摘要】：[目的] 利用合成抗原GST-HoxA1免疫BALB/c小鼠,制備針對HoxA1蛋白的單克隆抗體,通過Westen bloting檢測其特異性,FITC熒光素標記抗體,免疫熒光染色,研究其在HoxA1蛋白檢測中的應用價值。 [方法與結(jié)果] GST-HoxA1合成抗原免疫BALB/c小鼠,采用傳統(tǒng)單克隆抗體制備方法,通過細胞融合,HAT選擇,有限稀釋法克隆擴增,獲得穩(wěn)定分泌高純度抗HoxA1單克隆抗體的雜交瘤細胞株。利用小鼠體內(nèi)接種雜交瘤細胞制備含特異性抗體的腹水,通過辛酸—硫酸銨法純化,得到優(yōu)質(zhì)單克隆抗體。通過間接ELISA法測定抗體效價,Westen bloting檢測其的特異性。經(jīng)測定抗HoxA1單克隆抗體4D5效價為1:102400,Westen bloting結(jié)果發(fā)現(xiàn)4D5可以特異性結(jié)合靶細胞Hela中HoxA1蛋白,與載體蛋白GST無交叉反應;利用FITC熒光素標記抗體,免疫熒光染色結(jié)果顯示實驗組腫瘤細胞明顯熒光表達,對照組細胞僅見極微弱熒光表達。 [結(jié)論] 1.成功制備抗HoxA1單克隆抗體。 2.通過Westen bloting證實所制備單克隆抗體可與相應HoxA1蛋白特異性結(jié)合,用該抗體檢測Hela細胞裂解液,結(jié)果在相對分子質(zhì)量約36×10~3處有反應條帶;FITC熒光素標記抗體,免疫熒光染色檢測靶細胞Hela,證明該抗體可初步用于HoxA1蛋白檢測。
[Abstract]:[purpose] The monoclonal antibody against HoxA1 protein was prepared by immunizing BALB/c mice with synthetic antigen GST-HoxA1. The specific FITC fluorescein labeled antibody was detected by Westen bloting and immunofluorescence staining was used to study its application value in the detection of HoxA1 protein. [methods and results] GST-HoxA1 synthetic antigen was used to immunize BALB/c mice. The hybridoma cell lines secreting high purity monoclonal antibody against HoxA1 were obtained by using traditional monoclonal antibody preparation method, by cell fusion with hat selection and cloning by limited dilution method. Ascites containing specific antibodies were prepared by inoculating hybridoma cells in vivo and purified by octanoic acid-ammonium sulfate method to obtain high quality monoclonal antibodies. The antibody titer was determined by indirect ELISA method and the specificity of the antibody was detected by Westen bloting. The titer of anti HoxA1 monoclonal antibody 4D5 was 1: 102400 HoxA1 bloting. It was found that 4D5 could specifically bind HoxA1 protein in target cell Hela and had no cross reaction with carrier protein GST, and the antibody was labeled with FITC fluorescein. The results of immunofluorescence staining showed that the tumor cells in the experimental group showed obvious fluorescence expression, while in the control group, only very weak fluorescence expression was observed. [conclusion] 1. Monoclonal antibody against HoxA1 was successfully prepared. 2. It was confirmed by Westen bloting that the monoclonal antibody could specifically bind to the corresponding HoxA1 protein. The Hela cell lysate was detected by the antibody. The results showed that there were FITC-labeled antibodies at the relative molecular weight of 36 脳 10 ~ (-3). The detection of Hela by immunofluorescence staining showed that the antibody could be used for the detection of HoxA1 protein.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Preparation of monoclonal antibody against apoptosis-associated antigens of hepatoma cells by subtractive immunization[J];World Journal of Gastroenterology;2002年05期

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本文編號：1810570