重金属汞多克隆抗体的制备及间接竞争ELISA方法的研究

发布时间：2018-02-20 17:08

本文关键词： 重金属汞抗原多克隆抗体 ELISA　出处：《辽宁师范大学》2009年硕士论文　论文类型：学位论文

【摘要】： 本研究以制备汞的多克隆抗体为目的,将牛血清白蛋白(BSA)与汞离子通过谷胱甘肽偶联,合成了完全抗原Hg-GSH-BSA,将卵黄原蛋白(OVA)与汞离子通过青霉素G钠盐偶联,合成了完全抗原Hg-青霉素-OVA。以Hg-GSH-BSA作为免疫原,免疫新西兰大白兔一段时间后,采取动物的血清,用Hg-青霉素-OVA作为包被原,包被酶标板,采用间接ELISA法测定血清效价并对其进行鉴定,结果表明:采集到的血清中含有针对汞的抗体,血清效价为1∶204 800。在获得抗体的基础上,对运用抗体建立间接竞争ELISA检测的方法进行了初步探索,考察了ELISA检测方法的其中五个影响因子,确定的最佳检测条件为:包被抗原500倍稀释,抗血清12 000倍稀释,3%脱脂奶粉作为封闭剂,酶标二抗3000倍稀释,0.5mmol/L EDTA为螯合剂。以抑制率I为纵坐标,Hg2+浓度对数(lgC)为横坐标,建立检测曲线,得到线性回归方程为:Y= 0.1874X-0.0499,相关系数为0.9739,其中Y为抑制率,X为Hg2+浓度对数(lgC),检测限为0.0065mg/L。本研究成功制备了抗汞多克隆抗体,初步探索了汞的ELISA检测方法,为进一步的研究工作奠定了实验基础。
[Abstract]:In order to prepare polyclonal antibodies against mercury, bovine serum albumin (BSA) was coupled with mercury ion through glutathione to synthesize complete antigen Hg-GSH-BSA.The vitellogenin (OVA) was coupled with mercury ion through penicillin G sodium salt. The complete antigen Hg- penicillin OVA was synthesized. After immunizing New Zealand white rabbits with Hg-GSH-BSA for a period of time, the animal serum was taken, and Hg- penicillin-OVA was used as the envelope, and the enzyme labeled plate was coated with Hg- penicillin-OVA. Indirect ELISA method was used to determine the titer of serum and to identify it. The results showed that the collected serum contained antibodies against mercury, and the titer of serum was 1: 204,800. The method of indirect competitive ELISA detection with antibody was preliminarily explored. Five of the influencing factors of ELISA detection method were investigated. The best detection conditions were as follows: coated antigen was diluted 500 times. The antiserum 12 000 times diluted 3% defatted milk powder was used as the sealant, the enzyme labeled second antibody 3 000 times diluted with 0.5 mmol / L EDTA as the chelating agent, and the inhibition rate I as the logarithm of Hg2 concentration was taken as the horizontal coordinate, and the detection curve was established. The linear regression equation is:: y = 0.1874X-0.0499, the correlation coefficient is 0.9739, where Y is the inhibition rate X is the logarithm of Hg2 concentration, the detection limit is 0.0065 mg / L. In this study, polyclonal antibodies against mercury were successfully prepared, and the ELISA detection method of mercury was preliminarily explored. It lays the experimental foundation for further research work.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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