稻米中镉快速检测金标试纸条的研制

发布时间：2018-04-16 15:27

  本文选题：稻米 + 镉　； 参考：《长沙理工大学》2014年硕士论文

【摘要】：65%的中国人以稻米为绝对主食,2002年,农业部稻米及制品质量监督检验测试中心曾对全国市场稻米进行安全性抽检,结果显示,稻米中镉超标率为10.3%。未来中国农产品安全问题中,镉等重金属污染将取代农药,成为事故多发地带。传统的检测方法已不能满足需求,急需找到快速、灵敏、高效的新型检测方法,本论文以稻米中重金属镉为研究对象,以制备单克隆抗体为基础,进一步组装金标试纸条,以期建立现场、快速镉的免疫检测方法。采用大分子双功能螯合剂1-(4-异硫氰苄基)乙烯基二胺-N,N,N',N'-四乙酸(isothiocyano benzy-EDTA, iEDTA)螯合重金属镉离子形成六齿配合物,再分别偶联载体OVA和BSA,合成人工免疫抗原Cd-iEDTA-OVA和检测抗原Cd-iEDTA-BSA。石墨炉原子吸收分光光度计测得Cd-iEDTA-OVA和Cd-iEDTA-BSA中镉含量分别为174.6230μg-L-1和48.1881μg·L-1；紫外分光光度扫描结果表明完全抗原同时具备载体蛋白与半抗原的吸收特性,并测得OVA和BSA含量分别为1.7892mg-mL-/1和1.8065mg·mL-1,结果说明镉完全抗原合成成功,可用于后续试验研究。采用Cd-iEDTA-OVA多点少量注射BALB/c小鼠,获得抗血清,通过细胞融合、杂交瘤及克隆技术获得7F4和7E8两株特异性较好的杂交瘤细胞；通过体内诱生腹水法及辛酸-硫酸铵盐析、差量离心法纯化技术,获得7E8E5、7E8G9、7F488、7F4D6和7F4H85株单克隆抗体。酶联免疫吸附法(ELISA)结果表明,5株单抗均属IgGl型；亲和力均高,数量级达108；仅与Hg2+有较强交叉,与其他离子几乎无交叉；初步建立了Cd2+间接竞争ELISA法,最低检测限为260ng·mL-1,在260～5000ng·mL-1范围内趋近直线。SDS-PAGE法结果显示有且只有两条清晰的区带(重链带和轻链带),说明单抗纯度高。其中7E8E5和7E8G9效价相对高,分别达1：512000和256000,亲和常数也最高,分别为4.55×1081·mmoL-1和2.20×1081·moL-1,因此可从该两株单抗择优制备金标试纸条。采用柠檬酸三钠还原法制备13nm金颗粒,通过目测、可见-紫外分光光度法、透射电镜法鉴定,结果表明金溶液稳定于酒红色,且呈球形、大小均一,可用于标记7E8G9。通过条件优化得到：最佳标记pH为8.2,最小抗体用量为每1mL胶体金溶液中6.0μg单抗,T线包被浓度为1.0mg·mL-1,C线包被浓度为1.5mg·mL-1,且包被条件为37℃条件下干燥60min,最佳金标抗体稀释度为0.75倍,最佳封闭温度及时间为25℃干燥60min,最佳离子浓度为O.Olmol·L-1。将完全抗原Cd-iEDTA-BSA作为T线,羊抗鼠二抗作为C线,包被于硝酸纤维素膜上,纳米金颗粒标记的7E8G9单克隆抗体包被于金标垫上,组装成定性检测金标试纸条。试纸条的灵敏度为0.2 mg·kg-1、重复性良好、与Hg2+有较强交叉,最高共存浓度不得高于1.0mg·kg-1；与Zn2+有一定交叉,最高共存浓度不得高于10 mg·kg-1;与Cu2+、Fe2+、Ca2+、Mg2+、Al3+、.Pb2+几乎无交叉；贮存期约为1年。试纸条检测稻米中镉与GFAAS结果基本一致。
[Abstract]:65% of Chinese people take rice as the absolute staple food. In 2002, the quality Supervision and Test Center of Rice and its products of the Ministry of Agriculture carried out a sampling inspection on the safety of rice in the national market. The results showed that the cadmium excess rate in rice was 10.3%.In the future, cadmium and other heavy metal pollution will replace pesticides and become accident prone areas.The traditional detection method can not meet the demand, so it is urgent to find a new rapid, sensitive and efficient detection method. In this paper, the heavy metal cadmium in rice is taken as the research object, and the gold standard test strip is further assembled based on the preparation of monoclonal antibody.In order to establish a field, rapid immunoassay method for cadmium.Using macromolecular bifunctional chelating agent 1-chelating agent 1-chelate 4-isothiocyano benzy-EDTA (iEDTAA) to form hexadentate complexes, the artificial immune antigen (Cd-iEDTA-OVA) and the detection antigen (Cd-iEDTA-BSA) were synthesized by chelating the heavy metal cadmium ions. The artificial immune antigen (Cd-iEDTA-OVA) and the detection antigen (Cd-iEDTA-BSA) were synthesized by using the macromolecular bifunctional chelating agent 1-chelating agent 4-isothiocyano benzy-EDTA-EDTA-EDTA-EDTA-EDTA-EDTA.The content of cadmium in Cd-iEDTA-OVA and Cd-iEDTA-BSA was 174.6230 渭 g-L-1 and 48.1881 渭 g / L ~ (-1), respectively, by graphite furnace atomic absorption spectrophotometer.The contents of OVA and BSA were 1.7892mg-mL-/1 and 1.8065mg mL-1, respectively. The results showed that cadmium complete antigen was successfully synthesized and could be used for further study.The antiserum was obtained by injecting a small amount of Cd-iEDTA-OVA into BALB/c mice. Two hybridoma cell lines, 7F4 and 7E8, were obtained by cell fusion, hybridoma and cloning techniques, and ascites were induced in vivo and the octanoic acid-ammonium sulfate saltout was used.The monoclonal antibodies of 7E8E5O7E8G9, 7F488C7F4D6 and 7F4H85 strain were obtained by differential centrifugation.The results of Elisa showed that all of the 5 McAbs belonged to IgGl type, with high affinity and order of magnitude of 108. They only had strong cross with Hg2 and almost no cross with other ions. The indirect competitive ELISA method of Cd2 was established preliminarily.The lowest detection limit was 260ng mL-1. The results of the method showed that there were only two distinct bands (heavy chain band and light chain band) in the range of 260~5000ng mL-1. The results of SDS-PAGE showed that the purity of the McAb was high.The titer of 7E8E5 and 7E8G9 was relatively high, reaching 1: 512000 and 256000, respectively, and the affinity constant was the highest (4.55 脳 1081 mmoL-1 and 2.20 脳 1081 mol ~ (-1), respectively).13nm gold particles were prepared by tri-sodium citrate reduction method. The results showed that the gold solution was stable in wine red, spherical in size and uniform in size, and could be used to label 7E8G9 by visual measurement, visible ultraviolet spectrophotometry and transmission electron microscopy.The optimal labeling pH was 8.2, the minimum antibody dosage was 6.0 渭 g / 1mL colloidal gold solution and the concentration of 1.0mg mL-1C line coating was 1.5mg mL-1, and the best dilution of gold-labeled antibody was 0.75 times at 37 鈩，

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