Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods
Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods
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食品中丙烯酰胺检测的抗原合成策略和免疫分析方法开发
DOI：
10.1039/b716526a
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发表时间：
2008-01-01
期刊：
影响因子：
4.2
通讯作者：
Zhao, Meiping
中科院分区：
文献类型：
--
作者：
Zhou, Shuang;Zhang, Chen;Zhao, Meiping
丙烯酰胺是一种有毒和致癌的化合物，已被发现存在于一系列加工淀粉食品中。为了制备一种有效的丙烯酰胺免疫原复合物，将N-丙烯酰氧基琥珀酰亚胺（NAS）与牛血清白蛋白（BSA）以21.2：1的高摩尔比偶联。在常规方法后，通过用NAS-BSA缀合物的额外加强注射免疫兔获得抗血清。通过硫酸铵沉淀法纯化的IgG用BSA固定的免疫亲和柱进一步分级。收集的抗体和包被抗原（NAS-卵清蛋白）之间的亲和常数为6.7 × 10（7）L mol（-1）。天冬酰胺是食物中丙烯酰胺形成的关键前体，与抗体的交叉反应性可以忽略不计。建立了生物素-亲和素酶联免疫吸附试验（BA-ELISA），确定了BA-ELISA的最适反应介质为0.1molL（-1）NaHCO（3）（pH8.3，含0.5molL（-1）NaCl）。BA-ELISA的工作范围为10-100 000 ng/mL，检出限为6 ng/mL。将该方法应用于薯条和饼干中丙烯酰胺的检测，定量结果与高效液相色谱法的结果吻合良好。该方法对食品中丙烯酰胺的检测具有重要意义。
Acrylamide, a toxic and carcinogenic compound, has been found to be present in a range of processed starchy foods. To prepare an effective immunogen compound for acrylamide, N-acryloxysuccinimide (NAS) was conjugated to bovine serum albumin (BSA) at a high molar ratio of 21.2 : 1. Antisera were obtained by immunization of rabbits with additional booster injections of the NAS-BSA conjugate after the regular process. The IgGs purified by an ammonium sulfate precipitation method were further fractionated with a BSA-immobilized immunoaffinity column. The affinity constant between the collected antibody and coated antigen (NAS-ovalbumin) is found to be 6.7 x 10(7) L mol(-1). Asparagine, the key precursor of acrylamide formation in foods, showed negligible cross-reactivity to the antibody. A biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) was developed and the optimum assay medium was found to be 0.1 mol L(-1) NaHCO(3) (pH 8.3, containing 0.5 mol L(-1) NaCl). The BA-ELISA afforded a practical sensitivity with a working range of 10-100 000 ng mL(-1) and a detection limit of 6 ng mL(-1). The assay was applied to detect acrylamide in potato fries and biscuits and the quantitative results were in good agreement with those obtained by the high-performance liquid chromatography method. This immunoassay will be very useful for monitoring acrylamide in food samples.