呋喃妥因代谢物化学发光酶联免疫检测方法的研究

Study of Chemiluminescent Enzyme Immunoassay Method forNitrofurantoin Metabolite

关键词:  呋喃妥因代谢物  化学发光酶联免疫检测  包被原

Key words:  Nitrofurantoin Metabolite  CLEIA  Envelope antigen

摘要:

对呋喃妥因代谢物1-氨基乙内酰脲（AHD）进行半抗原改造，采用活化酯法将半抗原与卵清蛋白（OVA）偶联为包被原，与标准品竞争AHD单克隆抗体的抗原结合位点，加入辣根过氧化物酶标记羊抗鼠二抗，建立了AHD间接竞争化学发光酶联免疫(CLEIA)检测法，并对化学发光液、包被原与抗体最优稀释度、包被条件、封闭液和竞争时间五项参数进行优化。结果表明：该方法具有良好的特异性，IC50为0.753ng/mL，在鸡肉组织中的检测限为0.028μg/kg，添加回收率在80.54%~102.64%之间，变异系数均小于10%。与国标中液相色谱-串联质谱法（LC-MS/MS）和我国出入境行业标准中酶联免疫检测法（ELISA）相比，不仅降低了检测限，而且操作简便，为动物源性食品中呋喃妥因代谢物的残留提供了准确、便捷的分析检测手段，对严把食品安全质量关有重要参考价值。

Abstract:

The haptene of nitrofurantoin metabolite (AHD) was modified by hapten, then made it conjugate with ovalbmin(OVA) by using N-hyduoxysuccinimide ester method to synthesize envelope antigen. Then the compound was coupled to AHD monoclonal antibody competitively with standard reference. After the HRP labeled goat-anti-mouse secondary antibody coupled to the monoclonal antibody which conjugated with envelope antigen, an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for AHD was established. Then five test parameters, such as chemiluminescent solution, envelope antigen concentration and antibody concentration, enveloped condition, sealed liquid and competitive reaction time were optimized.The results indicated that the AHD CLEIA had good specificity for AHD.We obtained an IC50 value of 0.753ng/mL.We also obtained the limit of detection of 0.028μg/kg and recoveries rates of AHD ranged from 80.54%~102.64% in spiked chicken samples.The coefficient of variation was less than 10%.Compared with LC-MS/MS in national standards and ELISA in industry standard for CIQ, this method not only improve the detection limit ,but also operate easily. It could provide a convenient and accurate way for detection of AHD, which will have great value for the evaluation of food safety .