Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder
Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder
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荧光偏振免疫分析法检测牛奶和奶粉中三聚氰胺的开发
DOI：
10.1007/s00216-010-4599-2
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发表时间：
2011-02-01
影响因子：
4.3
通讯作者：
Lei, Hong-Tao
中科院分区：
文献类型：
--
作者：
Wang, Qiang;Haughey, Simon A.;Lei, Hong-Tao
建立了基于多克隆抗体的荧光偏振免疫分析法（FPIA）测定牛奶中三聚氰胺的方法。为了获得具有提高的灵敏度和特异性的抗体，6-肼基-1，3，5-三嗪-2，4-二胺与牛血清白蛋白偶联，并用作免疫原用于兔免疫。合成了三种具有不同结构和间隔桥的三聚氰胺荧光标记示踪剂。研究了示踪剂对测定特性的结构影响。6-（4，6-二氨基-1，3，5-三嗪-2-基氨基）-N-（2-（3-（3 '，6'-二羟基-3-氧代-2，3-二氢螺[茚-1，9 '-氧杂蒽]-5-基）硫脲基）乙基）己酰胺表现出比5-（2-（4，6-二氨基-1，3，5-三嗪-2-基）肼硫代甲酰胺基）-2-（6-羟基-3-氧代-3H-咕吨-9-基）苯甲酸和3-（4，6-二氨基-1，3，5-三嗪-2-基硫基）-N-（2-（3-（3 ′，6 ′-二羟基-3-氧代-3H-螺[异苯并呋喃-1，9 ′-咕吨]-5-基）硫脲基）乙基）丙酰胺。FPIA的检测限（10%抑制）为9.3 ng mL（-1），IC 50（50%抑制）值为164.7 ng mL（-1）。FPIA中的抗体对灭蝇杀虫剂环丙氨嗪显示出21.2%的交叉反应性，但对其他天然结构相关化合物没有交叉反应性。在加标牛奶和奶粉样品中测得的回收率范围为79.4%至119.0%。用该方法对添加三聚氰胺的牛奶样品进行了分析，并用高效液相色谱-质谱法进行了确证。结果表明，该方法可用于牛奶和奶粉中三聚氰胺残留量的检测。
A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3',6'-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL(-1) and the IC50 (50% inhibition) value was 164.7 ng mL(-1). The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography-mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.