高等学校化学学报

高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (1): 67.doi: 10.7503/cjcu20120346

• 分析化学 • 上一篇    下一篇

三聚氰胺单试剂荧光偏振免疫分析法

王强1, 黄启欣1, 孙远明1, 刘英菊2, 徐振林1, 雷红涛1   

  1. 1. 华南农业大学广东省食品质量安全重点实验室/农业部农产品贮藏质量安全风险评价重点实验室, 广州 510642;
    2. 华南农业大学理学院生物材料研究所, 广州 510642
  • 收稿日期:2012-04-13 发布日期:2012-12-31
  • 通讯作者: 雷红涛,男,博士,副教授,主要从事生物分析化学研究.E-mail:hongtao@scau.edu.cn E-mail:hongtao@scau.edu.cn
  • 基金资助:

    国家农业部行业公益项目(批准号: 201003008-08); 高等学校博士学科点专项科研基金优先发展领域项目(批准号: 20114404130002); 广州市科技计划项目(批准号: 12C12101663)和广东省高等学校成果转化项目(批准号: cgzhzd1005)资助.

Single-reagent Fluorescence Polarization Immunoassay for Melamine

WANG Qiang1, HUANG Qi-Xin1, SUN Yuan-Ming1, LIU Ying-Ju2, XU Zhen-Lin1, LEI Hong-Tao1   

  1. 1. Guangdong Provincial Key Laboratory of Food Quality and Safety/Key Risk Assessment Laboratory of Agricultural Product Preservation, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China;
    2. Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, China
  • Received:2012-04-13 Published:2012-12-31

PDF (PC)

被引次数

5

摘要：

用三聚氰胺半抗原(MEL)与异硫氰酸荧光素乙二胺(EDF)反应合成了荧光示踪物MEL-EDF, 示踪物与三聚氰胺抗体反应得到稳定的单试剂免疫复合物, 考察了其结合动力学和置换动力学过程, 建立了一种三聚氰胺单试剂荧光偏振免疫分析方法(SR-FPIA). 该方法的检出限(LOD)为3.2 ng/mL, 半抑制浓度(IC50)为157.1 ng/mL, 检测范围(IC20~IC80)为12.2~1012.4 ng/mL, 能够满足国家标准和国际食品法典委员会(CAC)对于食品中三聚氰胺最高残留限量的检测要求. 整个检测过程在15 min内完成, 并且单试剂免疫复合物在4℃下至少可稳定保存30 d以上. 本研究对开发三聚氰胺新型免疫检测方法具有重要意义.

关键词: 单试剂荧光偏振免疫分析, 示踪物, 三聚氰胺

Abstract:

Fluorescence-labeled melamine tracer(MEL-EDF) was synthesized via the reaction of melamine hapten(MEL) with 1-(2-aminoethyl)-3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thiourea(EDF). The tracer was then incubated with the antibody to prepare the single reagent immunocomplex, and both the binding and displacement kinetics were evaluated to obtain the equilibration time. Finally, a single reagent fluorescence polarization immunoassay(SR-FPIA) was developed to measure melamine. The SR-FPIA method demonstrated a detection limit(LOD) of 3.2 ng/mL, a 50% inhibition value(IC50) of 157.1 ng/mL and a working range(IC20-IC80) of 12.2-1012.4 ng/mL. The results indicated the proposed method can meet the detection requirement for the maximum residue level of melamine set by Chinese government and Codex Alimentarius Commission(CAC). The detection could be finished within 15 min, and the obtained single reagent immunocomplex was stable for more than 30 days at 4℃. This work will benefit to further development of SR-FPIA kit for melamine.

Key words: Single-reagent fluorescence polarization immunoassay, Tracer, Melamine

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