【作者】 牛涛；

【导师】 江天久；

【作者基本信息】 暨南大学 ， 环境科学， 2006， 硕士

【摘要】 常用的仪器型重金属检测方法在实际应用中存在诸多不便，不能满足当前重金属污染物快速检测的需要，开发快速、高效和低成本的重金属检测技术是对传统仪器型重金属检测方法的必要补充。 本论文根据我国重金属污染现状，选择环境中残留量较大的重金属Pb²⁺和Hg²⁺作为研究对象，试图开发出针对二者的快速检测技术。本论文包括以下两部分内容： 第一部分：重金属Pb²⁺单克隆抗体的制备。选择CHX-A"-DTPA作为双功能螯合剂，一方面螯合重金属Pb²⁺，另一方面通过其上的硫氰基团与载体蛋白KLH上的氨基偶联，从而制备出完全抗原Pb-CHX-A"-DTPA-KLH。用ICP-AES对此完全抗原含Pb²⁺量进行检测，其结果为38.02mg／L；在佐剂的辅助下免疫小鼠，333.33μg／只，免疫5次后，用间接ELISA方法检测免疫小鼠抗血清，其效价为1∶163840以上，并且含Pb²⁺抗原（Pb-CHX-A"-DTPA-BSA）检测OD值远大于无Pb（2+）抗原（CHX-A"-DTPA-BSA）；此后经过细胞融合、杂交瘤筛选以及克隆化等步骤，建立了分泌重金属Pb²⁺的单克隆抗体的细胞株1D4；其中细胞融合率（两块板）分别为51.04％和63.54％，杂交瘤细胞阳性率为2.04％；最后用小鼠体内诱生法生产抗Pb²⁺腹水型单抗，其效价为1∶204800，此腹水经饱和硫酸铵提纯后的蛋白质含量为19mg／ml。 第二部分：Hg²⁺的DTC螯合ELISA检测方法研究。利用二硫代氨基对Hg²⁺具有较高亲和力和ELISA信号放大原理，一方面在载体蛋白上构建二硫代氨基团，使之作为检测抗原包被在酶标板内；另一方面构建含Hg²⁺和酶的竞争抗原，其与待测样品中Hg²⁺竞争包被在酶标板内的二硫代氨基，运用竞争ELISA方法检测出样品中的Hg²⁺含量。本实验结果表明：此方法检测限为1ppb；具有较宽的pH检测（pH5.8～7.2）范围；离子强度对检测结果影响不大，只是在较高离子强度下（1M）有少许抑止；其它金属离子对其干扰较少(除Cu²⁺外)，可应用于环境样品Hg²⁺的快速检测。更多还原

【Abstract】 The methods of tranditionally instrument-intensive detection for heavy metals have many inconveniences in practice, and can’t satisfy the needs of the rapid detection, so the development of rapid, high-performance and low-cost heavy metals detection teniques is a necessary supplement for the tranditional detection.According to the status of heavy metal pollution in our country, the present study chose largely residual heavy metals Pb²⁺ and Hg²⁺ as research objects, and tried to develop the rapid detection techniques for the two metals.This paper consists of two parts as follows:Part One: The preparation of anti-Pb²⁺ monoclonal antibody （McAb）By selecting bifuntional chelator, the complete antigen （Pb-CHX-A"-DTPA-KLH） could be prepared by the chelator chelating Pb²⁺ and covalently linking itself to KLH. The concentration of Pb²⁺ in the complete antigen was 38.02mg/L; After 5 times’ mouse immunizations with the helps of freund’s complete and incomplete adjuvants, the antiserum of mouse was assessed by indirect ELISA, and the results showed the titer of antiserum was above 1 : 163840 and the OD values of Pb-loaded antigen（Pb-CHX-A"-DTPA-BSA） were much larger than those of metal-free antigen （CHX-A"-DTPA-BSA）; then synthesizing and secreting anti-Pb²⁺ McAb cell line 1D4 was obtained after a series of steps of cell fusion, hybridoma screening and positive hybridoma subcloning, etc; cell fusion rates of two 96-holes-culture plates were 51.04 % and 63.54 %, respetively; the positive rate of hybridoma cells was 2.04 %; At last, ascites fluids were produced in freund’s incomplete adjuvant-primed BALB/c mice by intraperitoneal injection of 1-2×10⁶ hybridoma cells, and its titer was above 1:204800 assessed by indirect ELISA, and the concentration of protein in ascites fluid purified by saturated ammonium sulfate was 19mg/ml. Part two: the method of DTC chelating ELISA detection for Hg²⁺The rapid analyse for Hg²⁺ was devised depending on the principles of dithiocarbamate’s higher avidity for Hg²⁺ and the signal amplification of ELISA. On one aspect, the detection antigen coated on the ELISA plate was constructed by dithiocarbamate bound to carrier protein （BSA）; On the other aspect, the competitive antigen contained Hg²⁺ and reporter enzyme （HRP） was synthesized, which is competitive with the Hg²⁺ in the environment samples for the dithiocarbamate coated on the ELISA plate; the concentrations of Hg²⁺ in samples could be obtained by更多还原