中文摘要
丙烯酰胺是近年来受到密切关注的热点食品加工污染物，其特点是分子结构简单、分子量小（71 Da），属于免疫分析领域很少涉足的极限分子量区域。本项目以丙烯酰胺作为主要目标物对这一问题进行了探索，详细研究了其全抗原合成和抗体制备的方法，通过与一系列分子量相近的结构类似物的对照，对其作为半抗原所必备的官能团进行了探索，设计了一种简单、高效的丙烯酰胺全抗原合成路线，成功制备了丙烯酰胺的多克隆和单克隆抗体。同时，利用所获得的抗体建立了丙烯酰胺的生物素-亲和素放大酶联免疫吸附分析方法，工作曲线范围10 ng/mL-500 μg/mL，检测限6.0 ng/mL，可用于食品和环境样品中丙烯酰胺的快速、高通量检测。项目还对丙烯酰胺与蛋白分子的相互作用机理进行了详细研究，为阐明丙烯酰胺的毒理作用提供了新的内容和丰富的实验依据。
英文摘要
Acrylamide has gained extensive international attention since it was discovered in a range of processed starchy foods. Due to its simple structure and small molecular weight, little has been reported on the development of immunoassays for acrylamide. In this project, a unique strategy was proposed to synthesize a complete antigen for acrylamide by using Nacryloxysuccinimide as the hapten instead of acrylamide itself, to ensure the preservation of the intact characteristic structure of acrylamide in the resultant conjugate, with a high molar ratio of 23:1 after optimization. Specific polyclonal and monoclonal antibodies towards acrylamide were both successfully obtained for the first time. The vinyl-carbonyl conjugate structure was found to be the key epitope of acrylamide for its relatively high immunogenicity as compared to other structurally similar compounds. The obtained antibodies were employed to establish a biotin-avidin amplified enzyme-linked immunosorbent assay (ELISA) for the determination of acrylamide. The working range and detection limit were found to be 10-100 000 ng/mL and 6 ng/mL, respectively.The assay was successfully applied to detect acrylamide in potato fries and biscuits. The interactions between acrylamide and proteins were carefully investigated by various approaches. The results of this project established a sound basis for further development of simple pretreatment procedures and rapid detection approach for acrylamide in foods. It is also very useful for intensive investigation of the toxicity of acrylamide in human body and taking effective measures to prevent and control the acrylamide contamination.
结题摘要
丙烯酰胺是近年来受到密切关注的热点食品加工污染物，其特点是分子结构简单、分子量小（71 Da），属于免疫分析领域很少涉足的极限分子量区域。本项目以丙烯酰胺作为主要目标物对这一问题进行了探索，详细研究了其全抗原合成和抗体制备的方法，通过与一系列分子量相近的结构类似物的对照，对其作为半抗原所必备的官能团进行了探索，设计了一种简单、高效的丙烯酰胺全抗原合成路线，成功制备了丙烯酰胺的多克隆和单克隆抗体。同时，利用所获得的抗体建立了丙烯酰胺的生物素-亲和素放大酶联免疫吸附分析方法，工作曲线范围10 ng/mL-500 μg/mL，检测限6.0 ng/mL，可用于食品和环境样品中丙烯酰胺的快速、高通量检测。项目还对丙烯酰胺与蛋白分子的相互作用机理进行了详细研究，为阐明丙烯酰胺的毒理作用提供了新的内容和丰富的实验依据。