测定食品中呋喃它酮代谢物3-氨基-5-吗啉甲基-2-恶唑烷酮的酶联免疫吸附法

Detection of furaltadone metabolite in food samples by an enzyme-linked immunosorbent assay

目的 建立以多克隆抗体为基础的测定食品中呋喃它酮代谢物3-氨基-5-吗啉甲基-2-恶唑烷酮(AMOZ)的间接竞争酶联免疫吸附分析法(ELISA).方法 以4-甲酰基苯氧基乙酸(4-FPA)为修饰剂,合成AMOZ半抗原衍生物,并使其分别与牛血清蛋白(BSA)和卵清蛋白(OVA)交联制备得到免疫原和包被抗原,经免疫动物(兔),获得抗NPAMOZ(AMOZ与衍生化试剂2-硝基苯甲醛所形成的结合物)的多克隆抗体.用酶联免疫分析法测定AMOZ(以NPAMOZ形式).结果 在包被抗原为2.5 ng/mL,抗体为1:300 000稀释,酶标二抗为1:10000稀释的优化条件下,ELISA测定AMOZ(以NPAMOZ形式)的IC50值(标准曲线中吸光度抑制至最大吸光度值50％时所对应的待测物浓度)为1.86 ng/mL.抗体与呋喃它酮的交叉反应率较高(70.7％),与AMOZ的交叉反应率仅为0.32％,与其余三种硝基呋喃抗生素及它们的代谢物以及其它八种药物的交叉反应率很小(＜0.01％),表明抗体的特异性高.四种市售的肉样(鱼肉、虾肉、鸡肉、猪肝)用作加标实验,加标浓度分别为0.5、1.0和5.0 ng/g,加标样品经衍生化处理后用ELISA测定,回收率为75.6％～112.2％,批间相对标准偏差(RSD)为8.3％～17.5％.结论 本法具有高灵敏性和高特特异性,能用于动物产品中AMOZ的检测.

Objective To establish a polyclonal antibody based indirect competitive enzyme-linked immunosorbent assay(ELISA) for the detection of furaltadone metabolite(3-amino-5-morpholinomethyl-2-oxazolidinone,AMOZ) in food samples.Methods With 4-formylphenoxy acetic acid(4-FPA) as a derivative reagent,carboxyphenyl AMOZ derivative(CPAMOZ) containing a carboxylic group at the end of the spacer was synthesized,which was then coupled to carrier proteins(bovine serum albumin or ovalbumin) for the production of immunogen and coating antigen.After immunizing animal(rabbit),polyclonal antibody against NPAMOZ(AMOZ nitrophenyl derivative) was produced.AMOZ(in the form of NPAMOZ) was detected by ELISA.Results Under optimal experimental conditions,e.g.coating antigen at 2.5 ng/mL,antibody diluted at 1:300,000,enzyme labeled second antibody diluted at 1:10,000,the sensitivity of the ELISA expressed by IC50 value(e.g.the analyte concentration at 50% inhibition of maximum signal) was found to be 1.86 ng/mL.The cross-reactivity(CR) of the antibody with furaltadone was 70.7%,while CR with AMOZ was only 0.32% and there was almost no CR with other three nitrofurans and their metabolites,as well as other eight tested drugs,indicating a high selectivity of the antibody.Four food samples,e.g.fish(carp),shrimp,chicken muscle and swine liver were spiked with AMOZ at the concentration of 0.5,1.0 and 5.0 ng/g.After derivatization,the formed NPAMOZ was analyzed by the proposed ELISA.The recovery and the relative standard deviation of the ELISA for spiked samples were found within 75.6%~112.2% and 8.3%~17.5%,respectively.Conclusion The method is sensitive and specific,and can be used for AMOZ detection in animal foods.