利用基因工程抗体建立新型双抗夹心ELISA法检测Cry1Ac毒素.docVIP

利用基因工程抗体建立新型双抗夹心ELISA法检测Cry1Ac毒素 张留圈1，2，张霄2，刘媛2，徐重新2，张存政2，谢雅晶2，寇莉萍1，刘贤金2 (1. 西北农林科技大学食品科学与工程学院，陕西 杨凌 712100；2. 江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地；农业部农产品质量安全控制技术与标准重点实验室；江苏省农业科学院食品质量安全与检测研究所，江苏 南京 210014) 摘要：为了利用基因工程抗体建立快速检测Cry1Ac毒素的新型双抗夹心ELISA方法，本试验分别用纯化后的抗Cry1Ac单链抗体和anti-Cry1Ac兔多克隆血清（Anti-Cry1Ac-PAbs）作为捕获抗体和检测抗体，通过方阵滴定确定其最佳工作浓度，建立双抗夹心ELISA法。然后用优化后的双抗夹心ELISA方法对检测Cry1Ac毒素的敏感性、特异性和回收率进行鉴定试验。结果表明，该方法最低检测限为0.91ng·mL -1，线性检测范围在1.04ng·mL -1~3.49μg·mL -1。5种稻米中Cry1Ac毒素的平均回收率为69.42%～87.95%，变异系数为1.19%～6.50%。因此，试验建立的双抗夹心ELISA法检测Cry1Ac毒素的敏感性、准确性和特异性均较良好，为检测转基因食品中Cry1Ac毒素提供了一种简便快速准确的方法。 Establishment of new Double-antibody Sandwich ELISA Method for Detection of Cry1Ac Toxin with Genetically Engineered Antibody ZHANG Liu-quan1,2, ZHANG Xiao2, LIU Yuan2, XU Chong-xin2, ZHANG Cun-zheng2, XIE Ya-jing2, KOU Li-ping1, LIU Xian-jin2, (1College of Food Science and Engineering,Northwest AF University,Yangling,Shaanxi 712100;2Key lab of Food Quality and Jiangsu Province-State Key Laboratary Breeding Base/Key Laboratary of Control Technology and Standard for Agro-product Safety and Quality, Institute of Food Safety,Jiangsu Academy of Agricultural Science,Nanjing ，jiangsu210014) Abstract: For the purpose of establishing a new double-antibody sandwich ELISA to detect Cry1Ac toxin rapidly with genetically engineered antibody, in the paper the purified anti-Cry1Ac single chain antibody was used as the capture antibody, and anti-Cry1Ac-PAbs was used as the detection antibody, their optimal working concentration was determined by titration phalanx. Sensitivity, specificity and recoveries were tested by the optimized double-antibody sandwich ELISA. The results showed that the limit of detection of Cry1Ac toxin was 0.91ng·mL -1, with the linear range from 1.04ng·mL -1 to 3.49μg·mL -1. The average recovery of Cry1Ac toxin in five types of rice was 69.42%～87.95%～6.50%Key words: Genetically engineered Antibody; The Double-antibody Sandwich ELISA; Cry1Ac toxin. 苏云金芽孢杆菌（Bacillus thuringiensis，简称Bt）是一种分布广泛