重金属离子单克隆抗体的制备及其新型免疫分析方法的研究

The exposure of a large amount of various heavy metals such as copper, mercury, cadmium, lead, etc. has resulted in a serious pollution to the environment upon which the human being living and society development are dependent. High heavy metal pollution also causes a big threat to the public health. The fundament to establish an immunoassay is the preparation of high quality antibody against target analyte. In this project, we first select an bifunctional reagent 1-(4-isothiocyanatebenzyl) ethylenediamine -N,N,N',N' - tetraacetic acid (ITCBE) for the preparation of immunogen, and then prepare the high specificity monoclonal antibody against copper and cadmium ions by the hybridoma technique. At optimal experimental conditions, the indirect competitive ELISA for copper and cadmium ions were established. In order to further improve the sensitivity or make the assay performance simpler, taking advantage of home-made mAb, we will develop ultrasensitive and specific competitive immunoassays for the detection of kinds of heavy metals based on surface plasmon resonance (SPR), and colloidal gold immunochromatography assay technique(CGICA) due to their advantages of free mark, high sensitivity and rapid. These novel methods with extremely high sensitivity was not only firstly established up to date for heavy metal ions, but also can be extended as an useful model for the detection of other heavy metals in environmental, pharmaceutical and food areas. In summary, this project was innovative and has high academic value.

快速定量检出环境及食品中重金属离子的研究已引起世界范围内的高度重视。本项目将首先选用双功能试剂1-(4-异硫氰苄基)乙烯基二胺-N,N,N',N'-四乙酸（ITCBE），制备铜离子和镉离子的免疫原，再利用细胞融合和杂交瘤技术获得抗铜离子和镉离子的单克隆抗体，建立检测两种重金属离子的间接竞争酶联免疫吸附分析方法（ELISA）。为进一步提高重金属离子检测方法的灵敏度并使测定更简捷，本项目组欲将免疫分析方法与实时在线、免标记的表面等离子体共振(SPR)技术和快速简便的胶体金免疫层析技术(CGICA)相结合，建立新型的检测重金属离子的免疫分析方法。其中多组分胶体金试剂条同时快速检测多种重金属离子的研究也为试剂条的市场化提供基础理论与技术支持。本项目的研究结果将为环境、药物和食品领域中重金属离子的快速痕量检测提供新的研究方法，具有较大的科学研究意义和实际应用价值。

本项目首先制备出了镉离子和铜离子的单克隆抗体，并分别建立了检测两种离子的间接竞争酶联免疫分析方法(Enzyme linked immunosorbent assay, ELISA)；然后，将抗体的强特异性与表面等离子体共振(Surface plasmon resonance, SPR) 的免标记、高灵敏度特点相结合，建立了检测镉离子的SPR竞争免疫分析方法；利用实验室先前制备出的特异性较好的汞离子和镉离子抗体，以硝酸纤维素膜为载体，以胶体金标记抗体作为检测探针，制备出了分别能检测镉离子和汞离子的胶体金免疫层析试纸条。同时，运用不同的荧光探针，构建了各种荧光生物传感器检测金属离子、甲胎蛋白、腺苷以及刀豆球蛋白A等；利用SPR技术具有免标记、可实时分析、灵敏度高等优点，结合适配体的特异性识别和结合能力，构建了一种新型生物传感器用于溶菌酶的高灵敏检测。此外，基于多种介孔二氧化硅（Mesoporous silica nanoparticles, MSNs）颗粒与表面活性剂脱氧胆酸钠（Sodium deoxycholate, NaDC）之间的相互作用，首次提出MSN-NaDC协同包合基质，并将该协同包合体系应用于RTP (Room temperature phosphorescence) 增敏基质；通过温度变化实验验证发现，上述MSN-NaDC协同包合体系的RTP诱导能力受温度变化影响较小，对磷光探针和多种多环芳烃的测定结果和分析性能都达到或高于传统分析手段，显示出良好的分析精确度和灵敏度。