Zhou Shuang, Zhang Chen, Wang Dan, Zhao Meiping
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.
Analyst. 2008 Jul;133(7):903-9. doi: 10.1039/b716526a. Epub 2008 Apr 3.
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.
丙烯酰胺是一种有毒且致癌的化合物，已被发现在一系列加工淀粉类食品中存在。为制备一种有效的丙烯酰胺免疫原化合物，将N-丙烯酰氧基琥珀酰亚胺（NAS）以21.2:1的高摩尔比与牛血清白蛋白（BSA）偶联。通过常规程序后用NAS-BSA偶联物额外加强免疫兔子来获得抗血清。通过硫酸铵沉淀法纯化的IgG再用固定化BSA的免疫亲和柱进行进一步分离。发现收集的抗体与包被抗原（NAS-卵清蛋白）之间的亲和常数为6.7×10⁷ L·mol⁻¹。食品中丙烯酰胺形成的关键前体天冬酰胺与该抗体的交叉反应可忽略不计。开发了一种生物素-抗生物素蛋白酶联免疫吸附测定法（BA-ELISA），发现最佳测定介质为0.1 mol·L⁻¹ NaHCO₃（pH 8.3，含0.5 mol·L⁻¹ NaCl）。该BA-ELISA具有实际灵敏度，工作范围为10 - 100,000 ng·mL⁻¹，检测限为6 ng·mL⁻¹。该测定法用于检测炸薯条和饼干中的丙烯酰胺，定量结果与高效液相色谱法获得的结果高度一致。这种免疫测定法对于监测食品样品中的丙烯酰胺将非常有用。
