化学发光酶免疫法测定动物源性食品中呋喃唑酮代谢物的含量

Determination of furazolidone metabolite in animal-derived food by chemiluminescence enzyme immunoassay

目的建立化学发光酶免疫法(chemiluminescent enzyme immunoassay, CLEIA)测定动物源食品中呋喃唑酮代谢物的分析方法。方法通过使用N-羟基琥珀酰亚胺酯法将衍生的CPAOZ半抗原与卵白蛋白(ovalbumin, OVA)缀合以合成包被抗原,优化CLEIA酶免疫方法的主要条件。对CLEIA酶免疫法的灵敏度、特异性、精密度及准确度进行评价,并就准确度指标同国家标准中的液相色谱串联质谱法进行对比分析。结果包被原和单克隆抗体稀释倍数分别为4000倍和1600倍, 1%脱脂乳封闭,竞争时间30 min, HRP-IgG孵育时间60 min时为最佳条件。CLEIA的线性方程为Y=-0.357X+1.459(r^2=0.984), IC50为0.486 ng/mL,线性范围为0.070～3.362 ng/mL。回收率在87.9%～92.6%之间,批内和批间变异系数分别为3.6%～7.5%和3.2%～6.3%。结论该方法简单,快速,可用于实验室或现场动物源性食品的AOZ筛选。

Objective To establish a method for the determination of furazolidone metabolites in animal-derived foods by chemiluminescent enzyme immunoassay(CLEIA).Methods The derivatized CPAOZ hapten was conjugated to ovalbumin(OVA)by the N-hydroxysuccinimide ester method to synthesize the coated antigen.The main conditions of the CLEIA enzyme immunization method were optimized.The sensitivity,specificity,precision and accuracy of the CLEIA enzyme immunoassay were evaluated,and the accuracy index was compared with the liquid chromatography tandem mass spectrometry in the national standard.Results The dilution ratios of the original and monoclonal antibodies were 4000 and 1600,respectively.The 1%skim milk was blocked,the competition time was 30 min,and the HRP-IgG incubation time was 60 min,which was the best condition.The linear equation for CLEIA was Y=-0.357 X+1.459(r^2=0.984)with IC50 of 0.486 ng/mL and a linear range of 0.070-3.362 ng/mL.The recovery rates ranged from 87.9%to 92.6%,and the intra-and inter-assay coefficients of variation were 3.6%-7.5%and 3.2%-6.3%,respectively.Conclusion This method is simple and rapid and can be used for AOZ screening of laboratory or on-site animal-derived foods.