间接竞争ELISA检测食品中的邻苯二甲酸二丁酯

Development of indirect Competitive ELISA for the detection of dibutyl phthalate in food

以4-氨基邻苯二甲酸为原料,通过酯化反应合成半抗原4-氨基邻苯二甲酸二丁酯,将合成的半抗原通过重氮化反应与钥孔血蓝蛋白(KLH)进行偶联制备免疫抗原,与牛血清白蛋白(BSA)进行偶联制备包被抗原。通过免疫新西兰大白兔获得邻苯二甲酸二丁酯抗血清,利用纯化获得的抗体和包被抗原建立邻苯二甲酸二丁酯间接竞争酶联免疫检测方法(ELISA)。该方法的IC50为40.68μg/L,最低检测限IC15为1.98μg/L,方法对邻苯二甲酸二丁酯具有较好的特异性。经对白酒、牛奶、食用油样品进行检测,添加回收率为84.57%~102.40%,用气相色谱-质谱法(GC-MS)进行验证,两种方法均呈现良好的线性相关性(R2=0.9908)。研究建立的方法可以被应用于食品中塑化剂邻苯二甲酸二丁酯的快速检测。

In this study, 4-aminophthalic acid was used to synthesize hapten Dibutyl 4-aminophthalate by esterification reaction, and the synthesized hapten was coupled with keyhole limpet hemocyanin (KLH) by diazotization reaction to prepare the immune antigen and coupled with bovine serum albumin (BSA) to prepare the coating antigen.The New Zealand white rabbits were immunized to obtain the antiserum against dibutyl phthalate.An indirect competitive enzyme-linked immunosorbent assay (ELISA) for dibutyl phthalate was established using the purified antibodies.For the developed ELISA, the IC 50 was 40.68 μg/L and the limit of detection was 1.98 μg/L.The method was applied to detect liquor, milk and edible oil samples, and the recoveries ranged from 84.57% to 102.40%.There was a good correlation ( R 2 =0.990 8) between the this ELISA and gas chromatography-mass spectrometry (GC-MS) for the detection of DBP in the spiked samples.The method established in this study can be applied to the rapid detection of dibutyl phthalate in food.