新型高灵敏赭曲霉毒素A间接竞争化学发光免疫分析法

A novel highly sensitive indirect competitive chemiluminescence enzyme-linked immunoassay for ochratoxin A

建立了一种测定赭曲霉毒素A的新型高灵敏化学发光间接竞争酶联免疫分析方法。以酶标赭曲霉毒素A二抗上的辣根过氧化物酶催化过氧化脲氧化3-(4-羟苯基)丙酸,生成具有荧光的3-(4-羟苯基)丙酸二聚体。并利用乙腈介质中双[2,4,6-三氯苯基]草酸酯和过氧化脲在增强剂咪唑的作用下反应产生强化学发光,以发光强度确定待检物中赭曲霉毒素A含量。结果表明,在最佳条件下IC50为0. 55 ng/m L,在0. 05～6. 08 ng/m L范围内有良好的线性关系,最低检出限为0. 01 ng/m L。样品加标回收实验显示葡萄干和葡萄汁样品的平均回收率分别为84. 55%～91. 36%和73. 32%～87. 64%,批内与批间变异系数均小于10%,精密度良好。该新型化学发光方法检测赭曲霉毒素A时发光强度更大、发光时间更长,可用于食品中赭曲霉毒素A的高灵敏度痕量检测。

A novel indirect highly sensitive competitive chemiluminescent immunoassay was established to determine ochratoxin A.Oxidation of 3-(4-hydroxyphenyl)propionic acid urea peroxide was catalyzed by horseradish peroxidase that was on an enzyme-labeled ochratoxin A secondary antibody to generate a fluorescent 3-(4-hydroxyphenyl)propionic acid dimer.A strong chemiluminescence was produced by the reaction between bis(2,4,6-trichlorophenyl)oxalate and urea peroxide in acetonitrile medium under the action of imidazole.The content of ochratoxin A in samples was determined by luminescence intensity.The results showed that under the optimal conditions,the IC 50 was 0.55 ng/mL with a good linearity in the range of 0.05-6.08 ng/mL,and a detection limit of 0.01 ng/mL.The average recovery rates of raisins and grape juice samples were 84.55%-91.36%and 73.32%-87.64%,respectively.The intra-assay and inter-assay variation coefficients were both less than 10%with good precision.This novel chemiluminescence method has higher luminescence intensity and longer emission time when detecting ochratoxin A.It can be used for detecting trace ochratoxin A in foods with high-sensitivity.