食品中苏丹红I酶联免疫检测方法的建立及酶学性质研究

Establishment of ELISA for Sudan I Residues in Food

DOI：10.3969/j.issn.1673-1689.2013.10.007

中文关键词: 苏丹红 酶免疫测定 多克隆抗体,辣椒粉

英文关键词: SudanⅠ ELISA polyclonal antibody chili powder

基金项目:国家“十二五”科技支撑项目(2012BAK17B10;2012BAK08B01)

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中文摘要:

为了检测食品中非法添加物苏丹红,采用重氮化法合成了2种苏丹红Ⅰ的衍生物,通过液相色谱-质谱(LC/MS)鉴定后,分别将两种衍生物与载体蛋白牛血清白蛋白(BSA)以及卵清蛋白(OVA)偶联制备完全抗原。紫外光谱表征结果证明衍生成功。将制备的BSA络合物做为免疫原免疫兔子,制备了多克隆抗体。采用方阵法确定了抗体和包被抗原的稀释比例。对影响酶免疫检测方法(ELISA)的因素包括包被液,封闭液,样品稀释液,抗体稀释液,反应时间等进行了优化。在最适反应条件下,以苏丹红质量浓度为横坐标,吸光度值为纵坐标建立了抑制曲线,线性范围为0.3~23.4 ng/mL,苏丹红Ⅰ的半数抑制率(IC50)为3.0 ng/mL,检测限(LOD)为0.1 ng/mL。交叉反应测试表明,与对位红交叉反应率为140%;与苏丹红Ⅱ、苏丹红Ⅲ、苏丹红G的交叉反应率分别为1.1%,6.85%,6.5%;与苏丹红Ⅳ的交叉反应率

英文摘要:

Two sudanI hapten derivatives were synthesized by diazotization and the resulting products were characterized by liquid chromatography-mass spectrum. The haptens were conjugated with bovine serum albumin(BSA)and ovalbumin(OVA) respectively. UV spectrum and electrophoresis confirmed the successful conjugation. The BSA conjugates were used to immunize New Zealand rabbits for preparation of antibody while OVA conjugates were used as coating antigen. Indirect competitive ELISA methods were developed. Various factors including coating buffer,blocking solution,sample solution,antibody solution,reaction time and so on were optimized.Under the optimum conditions,an inhibition curve was established with IC50value of 3.0 ng/mL and linear range 0.3~23.4 ng/mL. The limit of detection(LOD) was calculated as 0.1 ng/mL. Crossreaction tests showed that the antibody recognized para red well with cross-reactivity of 140% while little recognition to sudan Ⅱ(1.1%),sudan Ⅲ(6.85%),sudan G(6.5%) and sudan Ⅳ(less than0.1%). Fortified chili powder of two levels(5 ng/g and 10 ng/g) were tested using the developed immunoassay. The recoveries were 90.4% and 96.0% with coefficients of variation 1.8% and 4.9%.

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