重金属铅、镉单克隆抗体的制备及ELISA检测方法的建立

发布时间：2018-04-02 16:10

本文选题：铅　切入点：镉　出处：《吉林大学》2011年硕士论文

【摘要】：本研究通过双功能螯合剂将铅、镉离子分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,合成免疫抗原Pb-ITCBE-BSA、Cd-ITCBE-BSA和检测抗原Pb-ITCBE-OVA、Cd-ITCBE-OVA,采用SDS-PAGE电泳法和紫外分光光度法对制备的抗原进行鉴定。足垫注射方法免疫Balb/c小鼠,间接ELISA方法检测小鼠血清效价,采用PEG方法进行细胞融合,间接ELISA方法筛选阳性杂交瘤细胞,有限稀释法克隆杂交瘤细胞株,获得了抗铅离子的单克隆抗体细胞株3C1和抗镉单克隆抗体细胞株5E12。两株单抗细胞株的亚类均为IgG1,亲和力常数分别为1.19×108和1.02×108,其他金属离子的交叉反应均小于3%。通过体内诱生法制备腹水型单抗,采用亲和层析法纯化腹水。分别用3C1和5E12纯化的腹水建立优化了ELISA方法。采用间接竞争ELISA方法检测铅、镉标准品,并建立标准曲线,其中抗铅单克隆抗体的标准曲线回归方程是y=y=-16.325x+114.53,相关系数R2=0.9873,检测范围是20-160ng/mL,最低检测限为20ng/mL;抗镉单克隆抗体的标准曲线方程为y=-13.345+103.42,回归系数R2=0.994,检测范围是0.01-0.64μg/mL,最低检测限为0.01μg/mL。分别在纯净水、自来水和南湖水样中添加铅、镉标准品溶液,添加回收率分别为99.73%、101.01%、96.83%和101.62%、95.75%、98.49%。本研究为铅、镉离子酶联免疫快速检测试剂盒和免疫胶体金试纸条的开发利用奠定了一定的基础。
[Abstract]:In this study, lead and cadmium ions were coupled with bovine serum albumin (BSA) and ovalbumin (OVA) respectively to synthesize immune antigen Pb-ITCBE-BSA-Cd-ITCBE-BSA and detection antigen Pb-ITCBE-OVACd-ITCBE-OVA. the prepared antigens were identified by SDS-PAGE electrophoresis and UV spectrophotometry.Balb/c mice were immunized with foot pad injection method, serum titers of mice were detected by indirect ELISA method, cell fusion was performed by PEG method, positive hybridoma cells were screened by indirect ELISA method, hybridoma cell lines were cloned by limited dilution method.The monoclonal antibody cell line 3C1 and the monoclonal antibody cell line 5E12 were obtained.The affinity constants of the two McAb cell lines were 1.19 脳 10 ~ 8 and 1.02 脳 10 ~ 8, respectively. The cross reaction of other metal ions was less than 3.Ascites were purified by affinity chromatography.ELISA method was established by using 3C1 and 5E12 purified ascites.Indirect competitive ELISA method was used to detect lead and cadmium standard, and the standard curve was established.Lead and cadmium were added to pure water, tap water and Nanhu water, respectively. The recovery rates were 99.73 and 101.01% and 101.62% and 95.75%, respectively.This study laid a foundation for the development and utilization of the rapid detection kit for lead and cadmium ions and the immunocolloidal gold strip.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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