【摘要】：血液系統(tǒng)惡性腫瘤是當(dāng)今醫(yī)學(xué)上的頑癥之一,細(xì)胞膜表面分化抗原(CD)分子的研究能提高對(duì)其生物學(xué)行為的認(rèn)識(shí),有利于診斷治療。通過(guò)單克隆抗體(單抗)進(jìn)而研究細(xì)胞膜抗原生物學(xué)特性是蛋白質(zhì)功能研究的重要方法。ZCH(Zhejiang Childrens Hospital)-288a(簡(jiǎn)稱ZCH-288a)是本科研究室自行研制的鼠抗人造血細(xì)胞分化抗原的單抗,已經(jīng)提交第8屆國(guó)際人類白細(xì)胞分化抗原協(xié)作組會(huì)議(HLDA8)鑒定,根據(jù)HLDA8總部經(jīng)大量研究的反饋信息表明:該抗體識(shí)別的抗原性質(zhì)不明,屬國(guó)際上尚未認(rèn)識(shí)的血細(xì)胞膜新的分化抗原(新的CD分子)。因此,對(duì)其識(shí)別抗原編碼基因克隆與測(cè)序及其生物學(xué)功能研究有著重要的科學(xué)意義和潛在的應(yīng)用前景。 1 直標(biāo)鼠抗人新單抗ZCH-288a-FITC的研制及鑒定 為了深入研究ZCH-2B8a抗體識(shí)別抗原的表達(dá)譜及生物學(xué)功能,有利于利用流式細(xì)胞儀作多色分析和抗原阻滯試驗(yàn),減少實(shí)驗(yàn)誤差,擬將該抗體研制成異硫氰酸熒光素(Fluorescein isothiocyanate,FITC)直接標(biāo)記的抗體2B8a-FITC。在清潔級(jí)BALB/c小鼠腹腔內(nèi)注射1×10~6能分泌2BSa單抗(2B8aAb)的雜交瘤細(xì)胞,制備大量2B8aAb腹水。通過(guò)Econo-Pac蛋白A柱進(jìn)行親和層析,純化2B8aAb,純化后的2B8aAb經(jīng)SDS-PAGE凝膠電泳檢測(cè),純度高,達(dá)99%以上。采用改良Marsshall法,用FITC標(biāo)記純化的2B8aAb,多余FITC經(jīng)PBS充分透析除去,用紫外分光光度計(jì)測(cè)定A495/A280比值為0.56,介于FITC熒光抗體的理想比值0.3～1.0之間。流式細(xì)胞術(shù)檢測(cè)顯示,直標(biāo)單抗2B8a-FITC與間接標(biāo)記法2B8a+GAM-FITC在Raji細(xì)胞上的反應(yīng)峰型相似,雖然前者的平均熒光強(qiáng)度指數(shù)(MFI,98.61)略低于后者(106.89),但陽(yáng)性反應(yīng)率基本一致,分別為99.11%和99.00%,表明直標(biāo)熒光抗體2B8a-FITC制備成功。
[Abstract]:Malignant tumor of blood system is one of the intractable diseases in modern medicine. The study of membrane surface differentiation antigen (CD) molecule can improve the understanding of its biological behavior and be helpful for diagnosis and treatment. It is an important method to study the biological characteristics of cell membrane antigen by monoclonal antibody (McAb). ZCH (Zhejiang Childrens Hospital) -288a (ZCH-288a) is a mouse monoclonal antibody against human hematopoietic cell differentiation antigen developed by our laboratory. It was submitted to the 8th International Conference on Human Leukocyte differentiation Antigen (HLDA8) for identification. According to the feedback from a large number of studies at HLDA8 headquarters, the antigenic nature of the antibody was unknown. It is a new differentiation antigen (new CD molecule) of blood cell membrane that has not been recognized in the world. therefore The cloning and sequencing of its antigen-coding gene and the study of its biological function have important scientific significance and potential application prospect. 1 the direct labeled mouse anti-human monoclonal antibody ZCH-288a-FITC In order to study the expression profile and biological function of ZCH-2B8a antibody recognition antigen, It is helpful to use flow cytometry for polychromatic analysis and antigen block test to reduce the experimental error. It is proposed to prepare the antibody 2B8a-FITC labeled directly by fluorescein isothiocyanate (Fluorescein isothiocyanate,FITC). A large number of 2B8aAb ascites were prepared by intraperitoneal injection of 1 脳 10 ~ (-6) 2BSa monoclonal antibody (2B8aAb) into the peritoneal cavity of clean BALB/c mice. 2B8aAbwas purified by affinity chromatography on Econo-Pac protein A column. The purified 2B8aAb was detected by SDS-PAGE gel electrophoresis and its purity was over 99%. The modified Marsshall method was used to label the purified 2B8aAbby FITC. The excess FITC was fully dialyzed by PBS. The ratio of A495/A280 determined by UV spectrophotometer was 0.56, and the ideal ratio of FITC fluorescent antibody was between 0.3n.0. The results of flow cytometry showed that the peak pattern of direct labeled monoclonal antibody (2B8a-FITC) on Raji cells was similar to that of indirect labeled 2B8a GAM-FITC. Although the average fluorescence intensity index (MFI,98.61) of the former was slightly lower than that of the latter (106.89), the positive reaction rate was basically the same. They were 99.11% and 99.00%, respectively, indicating that the 2B8a-FITC was successfully prepared.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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