Cooper Kevin M, Samsonova Jeanne V, Plumpton Laura, Elliott Christopher T, Kennedy D Glenn
Queen's University Belfast, Department of Veterinary Science, Northern Ireland, UK.
Anal Chim Acta. 2007 May 29;592(1):64-71. doi: 10.1016/j.aca.2007.04.013. Epub 2007 Apr 19.
Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed--all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CCbeta) of 0.25 microg kg(-1) when a threshold of 0.21 microg kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 microg kg(-1) fortified sample, n=20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 microg kg(-1). NFZ-incurred muscles (12) containing SEM at 0.5-5.0 microg kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.
氨基脲（SEM）是被禁用的硝基呋喃类兽用抗生素呋喃西林（NFZ）的标记性残留，在食品中经常被检测到（近期欧盟硝基呋喃类快速预警中有47%涉及SEM）。然而，SEM作为NFZ的确定性标记物的有效性受到了其他来源产生的SEM的影响，这些来源包括偶氮二甲酰胺，一种塑料发泡剂和面粉处理添加剂。需要一种针对食品基质中SEM的廉价筛查方法——目前所有的SEM检测都使用昂贵的液相色谱-串联质谱仪。我们现在报告首次制备出针对衍生化SEM的抗体。一种新型的羧基苯基SEM衍生物被用于制备多克隆抗体，该抗体已被整合到一个半定量微量滴定板酶联免疫吸附测定（ELISA）中，并根据欧盟委员会第2002/657/EC号决定规定的标准进行了验证，可用于鸡肉检测。该抗体对衍生化SEM具有高度特异性，与NFZ的交叉反应率为1.7%，与多种其他硝基呋喃类药物和家禽药物的交叉反应可忽略不计。样品用邻硝基苯甲醛衍生化，同时进行蛋白酶消化，然后通过阳离子交换固相萃取进行提取。当应用0.21μg/kg的阈值来选择用于确证的样品时（最低观察到的加标样品为0.25μg/kg，n = 20），该ELISA对SEM的检测能力（CCβ）为0.25μg/kg，从而满足了欧盟硝基呋喃类药物1μg/kg的最低性能要求限值。通过液相色谱-串联质谱法检测出含有0.5 - 5.0μg/kg SEM的NFZ污染肌肉（12份），通过该ELISA方法检测全部呈阳性，该方法也适用于鸡蛋和鸡肝检测。
