3α-羟类固醇脱氢酶抗体的制备及标记
发布时间：2018-07-23 14:04
【摘要】： 睾丸酮丛毛单胞菌含有降解甾核的关键酶3α-羟类固醇脱氢酶(3α-HSD),通过包含许多酶的氧化甾核的复杂代谢途径,可以分解类固醇类物质。 本研究采用单克隆抗体技术制备抗3α-HSD单克隆抗体,获得了高表达抗3α-HSD的单克隆抗体的杂交瘤细胞株。主要方法是:将3α-HSD纯化后免疫雌性Balb/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经克隆和间接ELISA筛选,获得3株稳定分泌抗3α-HSD单克隆抗体的杂交瘤细胞株。经过鉴定:其腹水效价分别为1×10~6、1×10~5、1×10~4。 采用G蛋白亲和层析柱对腹水进行纯化,将纯化后的腹水单抗分别用辣根过氧化物酶(HRP)和异硫氰酸荧光素(FITC)标记,制备酶标抗体和荧光抗体,并对该酶标抗体和荧光抗体进行鉴定。制备所得酶标抗体和荧光抗体具有特异性强、敏感性高的特点。我们应用本研究制备的酶标单克隆抗体建立了双抗体夹心ELISA方法,对采集的水源样品进行检测。结果表明,该单克隆抗体显示了对类固醇类物质的高特异性,为建立科学完善的环境激素检测方法奠定了基础。
[Abstract]:The steroid degradation key enzyme 3 伪 -hydroxysteroid dehydrogenase (3 伪 -HSD) is found in Trichotoma tumega, which can decompose steroids through the complex metabolic pathway of oxidative steroid nucleus containing many enzymes. In this study, monoclonal antibodies against 3 伪 -HSD were prepared by monoclonal antibody technique, and hybridoma cell lines with high expression of anti 3 伪 -HSD monoclonal antibody were obtained. The main methods were as follows: female Balb/c mice were immunized with 3 伪 -HSD, and the spleen cells were fused with SP2/0 myeloma cells. By cloning and indirect ELISA screening, three hybridoma cell lines secreting monoclonal antibodies against 3 伪 -HSD were obtained. The ascites titers were 1 脳 10 ~ (6), 1 脳 10 ~ (5) and 1 脳 10 ~ (4), respectively. Ascites were purified by G protein affinity chromatography. The purified ascites McAbs were labeled with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), respectively. The enzyme labeled antibody and fluorescent antibody were identified. The enzyme labeled antibody and fluorescent antibody were prepared with strong specificity and high sensitivity. A double antibody sandwich ELISA method was developed for the detection of water source samples. The results showed that the monoclonal antibody showed a high specificity to steroids, which laid a foundation for the establishment of a scientific and perfect method for the detection of environmental hormones.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2139646
[Abstract]:The steroid degradation key enzyme 3 伪 -hydroxysteroid dehydrogenase (3 伪 -HSD) is found in Trichotoma tumega, which can decompose steroids through the complex metabolic pathway of oxidative steroid nucleus containing many enzymes. In this study, monoclonal antibodies against 3 伪 -HSD were prepared by monoclonal antibody technique, and hybridoma cell lines with high expression of anti 3 伪 -HSD monoclonal antibody were obtained. The main methods were as follows: female Balb/c mice were immunized with 3 伪 -HSD, and the spleen cells were fused with SP2/0 myeloma cells. By cloning and indirect ELISA screening, three hybridoma cell lines secreting monoclonal antibodies against 3 伪 -HSD were obtained. The ascites titers were 1 脳 10 ~ (6), 1 脳 10 ~ (5) and 1 脳 10 ~ (4), respectively. Ascites were purified by G protein affinity chromatography. The purified ascites McAbs were labeled with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), respectively. The enzyme labeled antibody and fluorescent antibody were identified. The enzyme labeled antibody and fluorescent antibody were prepared with strong specificity and high sensitivity. A double antibody sandwich ELISA method was developed for the detection of water source samples. The results showed that the monoclonal antibody showed a high specificity to steroids, which laid a foundation for the establishment of a scientific and perfect method for the detection of environmental hormones.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 王雅文;陈华林;王维;马帅;王一东;冀伟;;抗3α-HSD单克隆抗体制备方法的初步研究[J];长春理工大学学报(自然科学版);2013年05期
，本文编号：2139646
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