酱油与白酒中邻苯二甲酸二甲酯的化学发光酶免疫分析方法研究

Indirect Competitive Chemiluminescent Enzyme Immunoassay Method for Determination of Dimethyl Phthalate in Soy Sauce and Liquor

本研究以邻苯二甲酸二甲酯（ DMP）为研究目标,以4-氨基邻苯二甲酸二甲酯为半抗原,通过重氮化法偶联载体蛋白并免疫动物,制备针对DMP的特异性兔多克隆抗体。通过棋盘滴定法和单因素实验确定最佳的实验参数,即包被原浓度为50μg/L,一抗浓度为92.5μg/L,二抗浓度为1μg/mL,药物稀释液为pH 6.0的纯水,竞争反应时间为40 min,基于此建立了间接竞争化学发光酶联免疫分析法检测DMP。本方法对DMP的检出限为0.29μg/L,线性检测范围为0.74-30.32μg/L,与13种结构和功能类似物的交叉反应率均〈5%,通过倍比稀释降低白酒、酱油样品基质干扰,对样品的平均回收率在80.2%-116.0%之间,平均相对标准偏差〈3.6%,结果与GC-MS法的测定结果相符。本方法适用于食品样品中DMP的快速检测。

4-Amino dimethyl phthalate as the hapten was coupled to carrier protein and then used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate （ DMP） was thus obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme-linked immunoassay （ icCLEIA ） was developed. The experimental parameters of icCLEIA were optimized as follows： the concentration of coating antigen was 50 μg/L, the primary antibody concentration was 92. 5 μg/L, the secondary antibody concentration was 1μg/mL, distilled water （pH 6. 0） was used as diluent solution and the competitive reaction time was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0. 74μg/L to 30. 32μg/L with the limit of detection of 0. 29μg/L. The cross-reactivity of thirteen structural analogues was lower than 5%. The recovery of DMP from spiked liquor and soy sauce samples ranged from 80 . 2% to 116 . 0% and the average RSD was less than 3 . 6%. The detection results of the spiked liquor and soy sauce samples were consistent with those by standard gas chromatography-mass spectrometry method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.