土壤和植物产品中塑化剂的荧光偏振免疫分析方法研究

Phthalate Acid Esters (PAEs) are a diverse group of compounds that are broadly used in a wide array of industrial applications.PAEs may endanger the environment balance and affect the ontogenetic development of live organisms and their endocrine functions.PAEs have been detected throughout the environment include in soil in recent years.Exposure to PAEs in soil can be a stressor`for plants growing.So PAEs in the soil will affect the quality and safety of agricultural plant products. For quantitative analysis of residual PAEs in soil and plant products, the studying goals are to establish a rapid fluorescence polarization immunoassay method. A artifical conjugation of 4-nitro-phthalic acid with Bovine Serum Albumin (BSA) will be used as antigen through series of reaction such as esterification, reduction reactions and diazotization method. The artifical antigen will be used to immune mice, then by cell fusion and cloning screen, some monoclonal antibodies cell lines specific to Dibutyl phthalate（DBP）(also possible specific to DMP, DEP, DPP,DNPP, DNHP and DNOP) will be developed. The hapten molecules of DBP are coupled to quantum dots. Then a simple and efficient fluorescence polarization immunoassay will be established. The sensitivity, specificity, cross-reaction and recovery testing of the method are also being evaluated. The method may be used for determination of content of PAEs in the soil and for finding degradation method of PAEs and remedying contaminated soil. It can also be applied to detect concentration of PAEs in plant products, especially vegetables, fruits, and common food crops.This studies possess higher academic and application value for field soil quality improvement and plant products safety. It is also significant for reasonable use of the contaminated soil resources in the future.

土壤中塑化剂（Phthalic Acid Esters，PAEs）残留严重影响植物产品质量与安全，目前尚无快速准确的适应现场检测方法。以4-硝基邻苯二甲酸为前体物，通过酯化、还原化反应和重氮化方法将其与载体牛血清蛋白偶联并纯化后作为人工抗原。进而通过免疫动物、细胞融合与筛选，获得抗邻苯二甲酸二丁酯并抗邻苯二甲酸的二甲、乙、丙、戊、己、辛酯的单克隆抗体；以特定量子点与邻苯二甲酸二丁酯进行标记和纯化；最后建立一种简便快捷的PAEs荧光偏振免疫分析方法，对方法的灵敏度、特异性、交叉反应和回收率进行测试和评价。该方法可望应用于测定土壤中塑化剂的含量从而为土壤中塑化剂的降解和土壤修复寻求解决方法；也可以运用于植物产品特别是蔬菜、水果和常见粮食作物产品中塑化剂的检测。对于提高土壤质量与保障植物产品安全具有较高的学术和应用价值，甚至于将来更合理利用污染土壤资源也具有重大意义。

塑化剂是工业上广泛应用的增塑剂，有增强物质柔朝性的作用。主要用于聚氯乙稀塑料、食品包装袋、儿童玩具、香料、化妆品等的生产。同时塑化剂也是环境激素类污染物，它可以在一定条件下从塑料中通过溶出、迁移和排放等途径进入周围生态环境，甚至通过皮肤、呼吸系统和消化系统进入人体，进而对人体健康造成潜在危害。因此，建立一种快速检测塑化剂的方法具有十分重要的意义。.本文以4－硝基邻苯二甲酸为原料，分别与不同的醇反应衍生合成四种塑化剂结构类似物，包括直链结构、支链结构、连接臂和具有环状结构。半抗原偶联牛血清白蛋白BSA，制备完全人工免疫抗原DIBAP-BSA，免疫Balb/c小鼠，制备免疫脾细胞，与体外培养的骨髓瘤SP/20细胞融合，并经过间接ELISA检测筛选和有限稀释法亚克隆，得到阳性杂交瘤细胞株，并注射到不完全佐剂致敏的Balb/c小鼠腹腔，制备小鼠腹水，即单克隆抗体。单抗经过辛酸－硫酸铵法纯化，SDS-PAGE以及间接竞争ELISA方法鉴定，得到高特异性高灵敏度的DIBA单克隆抗体，并用于分析检测环境样品中DIBP残留量。.在这个实验中, 荧光素（FITC）与活化的5个DIBAP衍生物（不同的化学结构和间隔桥梁）氨基结合，得到荧光标记物，并通过薄层色谱(TLC)分离纯化和FPIA鉴定。与不同的标记物相比，DIBAP-FITC与DIBAP单克隆抗体结合度更好。通过标记物的浓度和单克隆抗体的稀释进一步评估提高荧光偏振免疫法(FPIA)的敏感性。通过快速、灵敏的FPIA方法检测菜心中的DIBP还未被报道，故在最优条件下，建立荧光偏振免疫分析法FPIA，用于检测菜心样品中DIBP的含量，线性范围为1.16-74.96 ng / mL，最低检测限为0.67 ng / mL，相关系数R2 = 0.9954。此外，其他6个同系化合物与DIBP的交叉反应率不到2.827%。并结合仪器方法HPLC和GC-MS对FPIA方法进行验证，三种方法同时对菜心样品做添加回收试验，FPIA的回收率为88.28－119.11 %，FPIA法与HPLC法的相似度为0.978，FPIA法与GC-MS法的相似度为0.980。由此数据可知，荧光偏振免疫分析方法的实验结果准确可靠，适用于分析检测样品中DIBP的残留量。