重金属镉离子抗原合成与单克隆抗体的制备

发布时间：2018-12-07 15:58

【摘要】： 随着人们生活水平的不断提高和环保意识的增强,对重金属污染尤为关注,残留检测急需加强。但常用的重金属检测方法需依靠大型仪器,大都存在样品处理繁琐、费用高、检测时间长等缺陷。本研究利用鳌合剂螯合Cd2+、偶联载体蛋白体外合成Cd2+免疫抗原和检测抗原;利用紫外分光光度法、三硝基苯磺酸法、以及抗血清效价检测对其进行评估;利用单克隆抗体制备技术初步建立镉离子的ELISA检测方法;旨在为重金属Cd~(2+)的残留检测提供一种方便、准确、特异、低廉、快速的新技术。结果:(1)合成抗原紫外吸收峰与载体蛋白紫外吸收峰有明显差异,游离氨基含量在33%～40%之间。(2)100μg/只剂量免疫小鼠后,抗血清效价明显高于50、200μg/只剂量免疫的小鼠,抗血清效价在1:81 920以上。(3)50%的PEG,SP2/0与小鼠脾细胞细胞比例为1:7.1时,融合率高达98.4%。(4)用BSA-IEDTA-Cd和BSA-IEDTA检测细胞上清,筛选出了2孔阳性杂交瘤细胞1A1和4E7,阳性率为0.35%;经3次阳性率100%的克隆后只获得了抗Cd2+的杂交瘤细胞株1A1。(5)利用已建立的间接竞争ELISA法检测发现,1A1细胞上清效价在1:1 024以上,腹水效价高于1:256 000。(6)腹水蛋白浓度为15.04 mg/mL,单克隆抗体为IgG1。结论:(1)IEDTA可以用于重金属Cd2+免疫抗原或检测抗原合成的螯合剂。(2)KLH作为免疫抗原的载体蛋白,免疫效果优于以BSA为载体蛋白;BSA用于检测抗原载体蛋白,可以降低成本,检测结果也相对稳定。(3)合理的免疫方案有助于产生高效价的抗血清,免疫效果与免疫剂量间无正相关性。(4)实验虽已制备出抗Cd2+的单克隆抗体,但细胞融合阳性较低,克隆不够稳定,抗原合成工艺尚需进一步优化。
[Abstract]:With the improvement of people's living standard and environmental protection consciousness, heavy metal pollution is paid more attention to, and the residue detection needs to be strengthened. However, the commonly used heavy metal detection methods rely on large scale instruments, most of them have the defects of complicated sample processing, high cost, long detection time and so on. In this study, Cd2 immune antigen and detection antigen were synthesized by chelating Cd2 and coupling carrier protein in vitro, and evaluated by UV spectrophotometry, trinitrobenzene sulfonic acid method and antiserum titer test. In order to provide a convenient, accurate, specific, inexpensive and rapid method for the detection of heavy metal Cd~ (2), a ELISA method for the detection of cadmium ion was established by using monoclonal antibody preparation technique. Results: (1) the ultraviolet absorption peak of synthetic antigen was significantly different from that of carrier protein, and the content of free amino group was between 33% and 40%. The titer of antiserum was significantly higher than that of mice immunized with a dose of 50200 渭 g / g. The titer of antiserum was above 1:81 920. (3) when the ratio of 50% PEG,SP2/0 to spleen cells was 1: 7.1, The fusion rate was as high as 98.4%. (4) the supernatant was detected by BSA-IEDTA-Cd and BSA-IEDTA, and 1A1 and 4E7 were screened out, the positive rate was 0.35. After three clones with 100% positive rate, only hybridoma cell line 1A1. (5) the titer of 1A1 cell supernatant was found to be above 1:1 by using the established indirect competitive ELISA assay. The titer of ascites was higher than 1: 256000. (6) the concentration of ascitic protein was 15.04 mg/mL, and monoclonal antibody was IgG1.. Conclusion: (1) IEDTA can be used as a chelating agent for heavy metal Cd2 immune antigen or antigen synthesis. (2) KLH as carrier protein of immune antigen is better than BSA as carrier protein. BSA can be used to detect antigen-carrier proteins, which can reduce the cost, and the results are relatively stable. (3) A reasonable immunization program helps to produce high titer antiserum. There was no positive correlation between immune effect and immune dose. (4) although monoclonal antibodies against Cd2 had been prepared, the fusion positive of cells was low, the clone was not stable, and the technology of antigen synthesis needed to be further optimized.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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