不同重金属双功能螯合剂对动物免疫应答的构效关系研究

In this study, four kinds of bifunctional chelating agents(e.g. Aminobenzyl-EDTA 、p- NH2-Bn-DTPA、p- NH2-Bn-NOTA and p- NH2-Bn-DOTA) and two kinds of heavy metal ions (e.g. Cu2+ and Pb2+ )were selected, then 8 kinds of chelate complexes were synthesized by Orthogonal Experimental Method and the stability constant of the complexes were detected respectively. After immunizing the New Zealand rabbits or BALB/C mice with the complete antigen which were conjugated with carrier protein such as BSA or OVA , the polyclonal antibodies and monoclonal antibodies were produced and purified. In order to clarify the structure-activity relationship between the bifunctional chelating agents and the ability of animal immune response, the multiple factor regression analysis method was developed for this research. In this method, many factors such as Structure Characteristic and stability constant of the chelate complexes, the heavy metal ionic spatial configuration in the different complexes and the parameter of affinity, specificity and sensitivity of the antibodies were selected and analysed. From this study, we hope to elicit the change regularity between the bifunctional chelating agents and the immunity recognition, and find out the mechanism which caused the differences. Finally ,Based on the pre-analysis, the enzyme-linked immunoassay(ELISA )for Cu2+ and Pb2+ in environmental and food samples would also been developed, which laid the foundation for developing the ELISA kits and standards in the future. Through the studies in the Project, it is significant in many ways , such as improve the synthetical efficiency for heavy metal complete antigen, perfect the immunoassay technical system, promote the development of heavy metal ELISA kits and standards, strengthen the supervision and management of heavy metal safety,and so on. Above all ,it is necessary and significant to carry out this research.

本项目以Aminobenzyl-EDTA 、p- NH2-Bn-DTPA、p- NH2-Bn-NOTA和p- NH2-Bn-DOTA等双功能螯合剂与Cu2+和Pb2+等重金属为研究对象，采用正交法制备8种螯合物并测定相应的稳定常数。与载体蛋白偶联制备完全抗原后免疫动物制备多克隆抗体和单克隆抗体，抗体纯化后进行抗体亲和力、特异性和灵敏度等特性参数测定，与双功能螯合剂的结构特征、螯合物稳定常数及重金属离子在螯合物中的模拟空间分布，进行多因素回归分析，建立双功能螯合剂对动物免疫应答的构效关系理论，初步阐明其差异性识别的形成机理，并在此基础上建立重金属在环境样品和食品中的ELISA分析技术，为下一步开展试剂盒产品开发和免疫检测技术标准提供依据。通过本项目实施，对于提高重金属完全抗原的制备技术，完善重金属免疫分析技术体系，推动重金属快速检测产品和技术标准的发展，加强重金属安全监管等方面均有重要意义。

1.分别将铜和铅离子与螯合剂EDTA、DTPA、DOTA、NOTA和载体蛋白偶联成功合成人工抗原并制备多克隆抗血清，效价测定表明用NOTA为螯合剂制备的抗原效果最佳。以Cu-NOTA-BSA和Pb-NOTA-BSA为免疫原，经过小鼠免疫、细胞融合、半固体培养基筛选、检测克隆等步骤，最终分别筛选到1株灵敏度高、特异性好的杂交瘤细胞株3B1和7B12，制备腹水并纯化获得单克隆抗体。.2.将抗铜和抗铅单克隆抗体分别进行包被抗原法和包被抗体法ELISA体系优化，重点对包被缓冲液、包被时间和温度、封闭液、离子强度、pH值等条件进行优化，筛选最佳免疫分析条件并进行水和贻贝样品的添加回收率试验。铜ELISA分析结果表明在包被抗原法模式下，水样平均添加回收率在81.1%～108.5%之间，变化范围在73%～131%之间，贻贝平均回收率在114.6%～123.9%之间，变化范围为93%～138%；在包被抗体模式下，水样平均添加回收率在76.3%～92.6%，变化范围在71%～104%之间，贻贝平均添加回收率在114.2%～127.6%之间，范围在96%～141%之间。铅ELISA结果表明在包被抗原法模式下水样平均添加回收率在79.4%～94.2%，变化范围在72.2%～105.7%之间，贻贝平均添加回收率在120.0%～123.9%之间，变化范围在103.2%～145.2%之间。在包被抗体法模式下水样平均添加回收率在78.4%～105.6%，变化范围在71.5%～112.1%之间，贻贝平均添加回收率在110.9%～133.2%之间，变化范围在94.4%～147.8%之间，结果表明整体上回收率属于可控范围。.3.分别对建立的铜和铅ELISA进行试剂盒的产品开发和试剂盒精密度和保存期进行试验。结果表明：从试剂盒保存期试验看，铜和铅的试剂盒不论是包被抗原法还是包被抗体法，其试剂盒的精密度比较均一，批内变异系数和批间变异系数均变化不大。从试剂盒保存期试验看，采用包被抗原法建立的试剂盒不论是铜还是铅，保存180 d后其OD450nm值才下降了20%左右，说明用该法建立的免疫分析方法比较稳定，试剂盒保存期在6个月左右；而采用包被抗体法建立的试剂盒则呈现出明显的递减效应，保存60 d后其OD450nm值下降20%左右，说明用该方法建立的试剂盒稳定性不佳，说明采用包被抗原法效果更佳。