重金属镉人工抗原构建及其多克隆抗体的制备

发布时间：2018-04-26 11:33

本文选题：镉 + X射线吸收精细结构光谱　；参考：《华中农业大学》2011年硕士论文

【摘要】：镉(Cadmium, Cd)是最常见的污染食品和饮料的重金属元素之一。Cd可以通过环境污染,生物浓缩和含Cd化肥的使用而导致食品和水体的污染。我国水稻、蔬菜等农作物Cd的检出率较高,超标现象严重。它具有较强的肾毒性、肝毒性、生殖毒性、免疫毒性、骨骼毒性、心脑血管毒性以及致癌等危害。目前,Cd的检测方法主要是仪器方法。该方法具有测量精度高,结果重复性好,特异性好,但大多需要借助大型仪器,成本较高,且前处理繁琐,不能满足现场、在线分析检测。而ELISA等免疫学方法,以其简单、快速、成本低且适应环境及市场的现场检测等优点受到广泛青睐。本研究采用双功能螯合剂p-SCN-Bn-DTPA构建了Cd离子的人工抗原,并采用紫外光谱法、三硝基苯磺酸法、原子吸收光谱法、聚丙烯酰胺凝胶电泳以及X射线吸收精细结构光谱对其进行分析鉴定。然后以构建成功的人工抗原免疫BALB/c小鼠,采用ELISA方法对抗血清的各项特性进行了研究,并采用间接竞争ELISA进行了加标回收和实际水样的检测,具体内容如下： 1镉离子人工抗原的构建 Cd是金属元素,分子量仅为112.4,它既没有反应原性,又没有免疫原性,只有通过双功能螫合剂与蛋白质等载体偶联后,才能刺激机体体产生抗体。本实验利用双功能螯合剂p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid,2-(4-异硫氰酸根苄基)-二乙烯三胺五乙酸)上的DTPA基团螯合Cd离子,并通过其SCN与载体蛋白牛血清白蛋白(bovine serum albumin, BSA)和卵清白蛋白(ovalbumin,OVA)的游离氨基偶联,制备得到免疫抗原Cd-p-SCN-Bn-DTPA-BSA (Cd-BSA)和包被抗原Cd-p-SCN-Bn-DTPA-OVA (Cd-OVA)。同时,为了分析Cd的偶联情况,还构建了p-SCN-Bn-DTPA-BSA (DTPA-BSA)、p-SCN-Bn-DTPA-OVA (DTPA-OVA)和Cd-DTPA。 2镉离子人工抗原的表征通过BSA、OVA、DTPA-BSA和DTPA-OVA的紫外光谱扫描分析,发现DTPA-BSA和DTPA-OVA的最大吸收波长较BSA和OVA发生了微弱的蓝移,分别是278 nm到270nm (BSA)和280 nm到268nm (OVA),它们的紫外扫描曲线也发生了改变。采用2,4,6-三硝基苯磺酸法(triniro-benzene-sulfonic-acid)法对Cd-BSA和Cd-OVA中游离氨基含量进行了测定,Cd-BSA中游离氨基数目为BSA的48.8%,而Cd-OVA的为OVA的41.4%。 SDS-PAGE结果表明,BSA (OVA)、DTPA-BSA (DTPA-OVA和Cd-BSA (Cd-OVA)的分子量是依次递增的。扩展X射线吸收精细结构光谱(extended X-ray absorption fine structure)结果表明,Cd-DTPA、Cd-BSA和Cd-OVA中Cd2+的配位类型及配位数基本相同。同时,它们的X射线吸收近边结构光谱(X-ray absorption near edge structure)结果表明,Cd-BSA和Cd-OVA中Cd2+的吸收边位置、形状、吸收峰强度均与Cd-DTPA的类似。吸收边位置相似表明,Cd-DTPA、Cd-BSA、Cd-OVA三种抗原中Cd离子的局部结构是一致的；吸收边形状相似,说明Cd-BSA和Cd-OVA与Cd-DTPA中Cd2+与DTPA形成螫合物的；吸收峰强度一致,则表明两种人工抗原中的Cd与Cd-DTPA中Cd一样均呈+2价。综上所述,人工抗原Cd-BSA和Cd-OVA构建成功。根据Cd-BSA和Cd-OVA的中游离氨基的替换率,结合它们的蛋白质含量,计算得出Cd-BSA和Cd-OVA中Cd2+与BSA和OVA的摩尔比分为28.7：1和11.7：1。 3抗镉离子抗血清的制备及其性质分析以Cd-BSA免疫三组BABL/c小鼠,每组三只。免疫周期均为首次免疫21d,后续加强免疫均为14d,免疫剂量依次为200、100和50μg/只/次。通过抗体产生进程曲线可以看出,三组BALB/c小鼠对Cd-BSA都能产生免疫应答,不同小鼠的免疫应答水平不一样,但总体的应答趋势基本一致。对三组小鼠第五次免疫后的抗血清进行性质分析,其效价从2.6×104到4.2x104不等,差异不大。经方阵滴定实验确定的三组小鼠抗原-抗体反应最佳工作条件后,采用间接竞争ELISA方法对三组小鼠抗血清进行分析,测得1号小鼠灵敏度为120ng/mL,3、4、5、6、7、8和9号小鼠灵敏度分别为200、70、59、58、155、150和167 ng/mL。选取三组小鼠中的1号、6号和8号小鼠抗血清进行特异性分析,结果显示,抗血清均对汞离子有一定的交叉反应,但是与铅、铁、铜、锌等其他参试金属离子没有交叉反应。 4镉离子ELISA方法的建立与湖水中镉离子的检测选取6号小鼠的抗血清为材料,通过间接竞争ELISA分析,建立了Cd2+的间接竞争ELISA方法。采用本方法,在自来水中添加Cd2+的浓度为10-200 ng/mL时,其线性方程为y=-20.62 x+87.069(R2=0.9919),加标回收率为85.3%-90.3%,平均为86.59%。从武汉市的三个湖泊中共采集3个样品进行Cd2+的ELISA,检测结果显示A湖中Cd2+浓度达到6.08ng/mL,与AAS分析结果的相符性为105.7%。
[Abstract]:Cadmium (Cd) is one of the most common heavy metals in contaminated food and beverages..Cd can cause food and water pollution through environmental pollution, biological concentration and the use of Cd chemical fertilizer. The detection rate of Cd in rice and vegetables in China is high and the phenomenon is serious. It has strong renal toxicity, hepatotoxicity, reproductive toxicity and immunity. At present, the method of detection of Cd is mainly instrument method. This method has high precision, good repeatability and good specificity, but most of them need large instrument, and the cost is high, and the preprocessing is tedious. It can not meet the site, on-line analysis and detection. And ELISA and other immunological parties The method is widely favored because of its simple, fast, low cost and adaptability to environmental and market site detection.
In this study, a double functional chelating agent p-SCN-Bn-DTPA was used to construct an artificial antigen of Cd ion, and it was identified by UV spectrometry, three nitrobenzene sulfonic acid method, atomic absorption spectrometry, polyacrylamide gel electrophoresis and X ray absorption fine structure spectrum. Then, a successful artificial antigen was used to immunize BALB/c mice and to use E. LISA method was used to study the characteristics of anti serum, and indirect competitive ELISA was used for the recovery of standard addition and the detection of actual water samples.
Construction of 1 cadmium ion artificial antigen
Cd is a metal element with a molecular weight of only 112.4. It does not respond to the original and immunogenicity. Only through the coupling of the double functional chelating agent to the protein and other carriers can the body produce antibodies. This experiment uses the bifunctional chelating agent p-SCN-Bn-DTPA (2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid, 2- (4- different). DTPA group of thiocyanate benzyl) - two ethylene three amine five acetic acid chelate Cd ion, and through its SCN and carrier protein bovine serum albumin (bovine serum albumin, BSA) and ovalbumin (ovalbumin, OVA) free amino coupling, preparation of immune antigen Cd-p-SCN-Bn-DTPA-BSA (Cd-BSA) and inclusion antigen Cd-p-SCN-Bn-DTPA-OVA. OVA. At the same time, in order to analyze the coupling of Cd, p-SCN-Bn-DTPA-BSA (DTPA-BSA), p-SCN-Bn-DTPA-OVA (DTPA-OVA) and Cd-DTPA. were also constructed.
Characterization of 2 cadmium ion artificial antigen
Through the UV spectrum scanning analysis of BSA, OVA, DTPA-BSA and DTPA-OVA, it was found that the maximum absorption wavelength of DTPA-BSA and DTPA-OVA had a weak blue shift than that of BSA and OVA, which were 278 nm to 270nm (BSA) and 280 nm, and their UV scanning curves were also changed.
The content of free amino groups in Cd-BSA and Cd-OVA was determined by 2,4,6- three nitrobenzene sulfonic acid method (triniro-benzene-sulfonic-acid). The number of free amino groups in Cd-BSA was 48.8% of BSA, and Cd-OVA was 41.4%. of OVA.
SDS-PAGE results show that the molecular weights of BSA (OVA) and DTPA-BSA (DTPA-OVA and Cd-BSA (Cd-OVA) are increasing sequentially.
The extended X ray absorption fine structure spectra (extended X-ray absorption fine structure) results show that the coordination type and coordination number of Cd2+ in Cd-DTPA, Cd-BSA and Cd-OVA are basically the same. The placement, shape, and absorption peak intensity are similar to that of Cd-DTPA. The absorption edge position similarity indicates that the local structure of the Cd ions in the three antigens of Cd-DTPA, Cd-BSA and Cd-OVA is the same; the shape of the absorption edge is similar, indicating that Cd-BSA and Cd-OVA are formed by Cd2+ and DTPA in Cd-DTPA, and the peak intensity of the absorption peak is consistent with that of the two kinds of artificial antigens. Cd in Cd-DTPA is the same as +2 price.
In conclusion, artificial antigens Cd-BSA and Cd-OVA have been successfully constructed.
According to the substitution rate of the middle reaches of Cd-BSA and Cd-OVA and the content of their protein, it is calculated that the molar ratio of Cd2+ to BSA and OVA in Cd-BSA and Cd-OVA is 28.7:1 and 11.7:1..
Preparation and characterization of 3 anti cadmium antiserum
Three groups of BABL/c mice were immunized with Cd-BSA, with three mice in each group. The immune cycle was 21d for the first time, and the subsequent enhanced immunization was 14d. The immunization dose was 200100 and 50 mu g/ only. The immune response of three groups of BALB/c mice could be produced by the antibody production process curve. The immune response level of different mice was different, but the total immune response of different mice was different, but the total number of immune responses in different mice was different. The response rate of the body was basically the same. The antisera of the three groups of mice were analyzed after fifth times of immunization, and their titers ranged from 2.6 * 104 to 4.2x104, with little difference.
After the best working conditions of three mice antigen antibody reaction determined by the square titration experiment, three groups of mice were analyzed by indirect competitive ELISA method. The sensitivity of No. 1 mice was 120ng/mL, and the sensitivity of 3,4,5,6,7,8 and 9 mice was 200,70,59,58155150 and 167 ng/mL. respectively, 1, 6 and 8 of the mice were selected. The specific analysis of antiserum in mice showed that the antiserum had a certain cross reaction to the mercury ion, but there was no cross reaction with other metal ions, such as lead, iron, copper and zinc.
4 establishment of cadmium ion ELISA method and detection of cadmium ion in lake water
Using the antiserum of No. 6 mice as material, the indirect competitive ELISA method of Cd2+ was established by indirect competitive ELISA analysis. The linear equation of Cd2+ was y=-20.62 x+87.069 (R2=0.9919) and the recovery rate was 85.3%-90.3%, and the average of 86.59%. from three lakes in Wuhan. 3 samples were collected for ELISA of Cd2+. The results showed that the Cd2+ concentration in A Lake reached 6.08ng/mL, and the consistency with AAS analysis was 105.7%..

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【相似文献】