一个新的白细胞膜抗原ZCH-2B8a编码基因及其生物学特性的研究
发布时间：2018-09-12 06:43
【摘要】：血液系统恶性肿瘤是当今医学上的顽症之一,细胞膜表面分化抗原(CD)分子的研究能提高对其生物学行为的认识,有利于诊断治疗。通过单克隆抗体(单抗)进而研究细胞膜抗原生物学特性是蛋白质功能研究的重要方法。ZCH(Zhejiang Childrens Hospital)-288a(简称ZCH-288a)是本科研究室自行研制的鼠抗人造血细胞分化抗原的单抗,已经提交第8届国际人类白细胞分化抗原协作组会议(HLDA8)鉴定,根据HLDA8总部经大量研究的反馈信息表明:该抗体识别的抗原性质不明,属国际上尚未认识的血细胞膜新的分化抗原(新的CD分子)。因此,对其识别抗原编码基因克隆与测序及其生物学功能研究有着重要的科学意义和潜在的应用前景。 1 直标鼠抗人新单抗ZCH-288a-FITC的研制及鉴定 为了深入研究ZCH-2B8a抗体识别抗原的表达谱及生物学功能,有利于利用流式细胞仪作多色分析和抗原阻滞试验,减少实验误差,拟将该抗体研制成异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)直接标记的抗体2B8a-FITC。在清洁级BALB/c小鼠腹腔内注射1×10~6能分泌2BSa单抗(2B8aAb)的杂交瘤细胞,制备大量2B8aAb腹水。通过Econo-Pac蛋白A柱进行亲和层析,纯化2B8aAb,纯化后的2B8aAb经SDS-PAGE凝胶电泳检测,纯度高,达99%以上。采用改良Marsshall法,用FITC标记纯化的2B8aAb,多余FITC经PBS充分透析除去,用紫外分光光度计测定A495/A280比值为0.56,介于FITC荧光抗体的理想比值0.3～1.0之间。流式细胞术检测显示,直标单抗2B8a-FITC与间接标记法2B8a+GAM-FITC在Raji细胞上的反应峰型相似,虽然前者的平均荧光强度指数(MFI,98.61)略低于后者(106.89),但阳性反应率基本一致,分别为99.11%和99.00%,表明直标荧光抗体2B8a-FITC制备成功。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
本文编号：2238212
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
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