荧光偏振免疫法测定水产品中的丁香酚

A fluorescence polarization immunoassay for the detection of eugenol residues in aquatic products

将丁香酚(Eul)与丙烯酸甲酯进行衍生化反应,合成了一种新型的丁香酚半抗原4-(4羟基-3-甲氨基苯基)-丁-2-烯酸(Eul-Aca)。采用活性酯法将丁香酚半抗原与乙二胺异硫氰酸荧光素(EDF)偶联,分别制备了同源和异源荧光示踪物。通过比较不同示踪物的抗体稀释度和灵敏度,优化反应时间等检测条件,建立了一种检测水产品中丁香酚的荧光偏振免疫分析法(FPIA)。结果表明:异源示踪物(Eul-AcaEDF)具有更优的检测灵敏度,抗体稀释度为1/200,反应时间仅需5 min。该方法的半抑制浓度(IC_(50))为11.2μg/L,检测线性范围为1.1~111.5μg/L,检出限(LOD)为0.24μg/L。实际样品中丁香酚的加标回收率为80.6%~107.4%,相对标准偏差(RSD)<15%。FPIA检测结果与气相色谱-质谱(GC-MS)法结果具有良好的一致性(r=0.993),适用于水产品中丁香酚的残留检测。

Eugenol was derivatized with methyl acrylate to obtain 4-(4-hydroxy-3-methamphenyl)-butyl-2-acrylic acid.The hapten was coupled to fluoresceinthiocarbamyl ethylenediamine(EDF)using the active ester method to produce homologous and heterologous tracers.The influence of tracer structures on assay parameters such as antibody dilution and incubation time were investigated.Furthermore,a fluorescence polarization immunoassay(FPIA)was developed to detect eugenol residues in aquatic products.The results showed that the heterologous tracer(Eul-Aca-EDF)exhibited higher sensitivity with an antibody titer of 1/200.The reaction time of FPIA was only 5 min.The detection limit of eugenol,the IC_(50) value,and the working range were 0.24,11.2 and 1.1-111.5μg/L,respectively.The recoveries of spiked aquatic products were in the range of 80.6%-107.4%,with the relative standard deviations(RSDs)less than 15%.The FPIA also showed a good correlation to the GC-MS method(r=0.993).Therefore,the developed FPIA exhibited potential practicality in the detection of eugenol residues in aquatic products.