丁草胺荧光偏振免疫分析示踪物的合成与鉴定.pdfVIP

中国农业科学 2008,41(11):3651-3655 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2008.11.027 丁草胺荧光偏振免疫分析——示踪物的合成与鉴定 薛 钢，雷红涛，沈玉栋，刘细霞，吴 青，孙远明，王 弘 （华南农业大学食品质量安全研究所/广东省食品质量安全重点实验室，广州 510642 ） 摘要：【目的】为建立除草剂丁草胺的荧光偏振免疫检测方法，对关键试剂示踪物进行合成与鉴定。【方法】 以异硫氰酸荧光素、乙二胺和己二胺为原料，合成了异硫氰酸荧光素乙二胺 （EDF）和异硫氰酸荧光素己二胺（HDF） 两种中间体，薄层层析法分离出目标产物，电喷雾电离质谱法鉴定；然后将 5-氨基荧光素（AF）、EDF、HDF 分别 与丁草胺半抗原（BMPA）连接，得到3种不同间隔臂的示踪物（BMPA-AF，BMPA-EDF和BMPA-HDF），利用抗血清稀 释曲线测定抗血清效价，竞争抑制曲线测定不同示踪物检测灵敏度。【结果】3种示踪物合成成功，抗血清稀释曲 线和竞争抑制曲线显示 3 种示踪物的抗血清效价分别为 1/600、1/1500 和 1/600，半抑制浓度分别为 1 292、433 和298 ng·ml-1 。【结论】合成出3种不同间隔臂的丁草胺示踪物，对示踪物的性质进行初步研究，为下一步建立丁 草胺荧光偏振免疫分析方法奠定了基础。 关键词：丁草胺；荧光偏振免疫分析；示踪物；异硫氰酸荧光素 Development of Fluorescence Polarization Immunoassay for the Herbicide Butachlor—Synthesis and Identification of Tracers XUE Gang, LEI Hong-tao, SHEN Yu-dong, LIU Xi-xia, WU Qing, SUN Yuan-ming, WANG Hong (Institute of Food Quality and Safety, South China Agricultural University/Guangdong Key Lab of Food Quality and Safety, Guangzhou 510642) Abstract: 【Objective 】To establish fluorescence polarization immunoassay method (FPIA) for the herbicide butachlor, the key reagent (tracer) was synthesized and identified. 【Method 】Using fluorescein isothiocyanate (FITC), ethylenediamine and hexylenediamine as materials, fluoresceinthiocarbamyl ethylenediamine (EDF) and fluoresceinthiocarbamyl hexylenediamine (HDF) were obtained, which were purified by thin layer chromatography and identified by ESI-MS. Various tracers (BMPA-AF, BMPA-EDF and BMPA-HDF) with different lengthes of bridge between the hapten molecule and 5-Aminofluorescein (AF), EDF, HDF, were synthesized to achieve high-sensitivity FPIA. The titers of antiserum and the sensitivity of various trace