一种检测杂环胺类化合物的酶联免疫检测方法的建立

为了制备一种广谱性杂环胺抗体,并建立一种可以实现多种杂环胺同时检测的快速分析方法。以杂环胺2-氨基-3,4-二甲基咪唑4,5-f]喹喔啉(MeIQx)为原料,将其与丁二酸单甲酯酰氯(MCO)反应合成杂环胺半抗原,通过活化酯法将半抗原与蛋白偶联制备免疫原进一步制备多克隆抗体,最终建立间接竞争酶联免疫分析方法(ic-ELISA)。该方法灵敏度(IC₅₀,以MeIQx计)为81.16 μg/L,检测限(IC₁₅)为12.07 μg/L。方法对喹啉类杂环胺(IQ、MeIQ)、喹喔啉类杂环胺(IQx、MeIQx、4,8-DiMeIQx、7,8-DiMeIQx、4,7,8-TriMeIQx)以及吡啶类杂环胺(PhIP)具有相同的识别能力,交叉反应率均达到93%以上。油炸牛肉和肉松样品中杂环胺(MeIQx)添加回收率在91.18%~98.64%之间,检测结果与液相色谱串联质谱法(LC-MS/MS)有很好的一致性(R2=0.9927)。本文建立的酶联免疫分析方法可以实现喹啉类、喹喔啉类以及吡啶类杂环胺总量的检测,为热加工肉制品中杂环胺的检测提供了一种简单、准确的快速检测方法。

Establishment of an Enzyme-Linked Immunosorbent Assay Method for Detection of Heterocyclic Amines

A broad-spectrum heterocyclic aromatic amine polyclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)was developed to simultaneously detect multiple heterocyclic aromatic amines. MeIQx was applied to synthesize the hapten via reacting with monomethyl succinate chloride(MCO). The polyclonal antibody was prepared by coupling hapten to protein by activated ester method to prepare immunogen,and the ic-ELISA method was established finally. The sensitivity of this ELISA(IC₅₀,for MeIQx)was 81.16 μg/L and the limit of detection(IC₁₅)was 12.07 μg/L. This method had similar recognition capacity for the quinoline heterocyclic aromatic amines(IQ,MeIQ),quinoxaline heterocyclic aromatic amines(IQx,MeIQx,4,8-DiMeIQx,7,8-DiMeIQx,4,7,8-TriMeIQx)and pyridines(PhIP)and the cross-reaction rates reached more than 93%.The recoveries of heterocyclic aromatic amine(MeIQx)in fried beef and dried meat floss samples were between 91.18% and 98.64%. There was a good correlation(R2=0.9927)between the ELISA and liquid chromatography-tandem mass spectrometry(LC-MS/MS)for the detection of MeIQx in the spiked samples. The ELISA established in this paper can be applied to detect the total amount of quinoline,quinoxaline and pyridine heterocyclic aromatic amine,and provide a simple and accurate approach for the detection of multiple heterocyclic aromatic amines in processed meat products.