间接竞争酶联免疫法测定鱼体中的亚甲基蓝  

Detection of methylene blue in fish by indirect competitive enzyme linked immunosorbent assay

作　　者：黄宣运[1,2,3,4] 杨光昕 孔聪 黄冬梅[1,2] 蔡友琼 HUANG Xuan-Yun;YANG Guang-Xin;KONG Cong;HUANG Dong-Mei;CAI You-Qiong(East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Shanghai 200090,China;Laboratory of Quality&Safety Risk Assessment for Aquatic Products(Shanghai),Ministry of Agriculture and Rural Affairs,Shanghai 200090,China;Centre for Research on Environmental Ecology and Fish Nutrition of the Ministry of Agriculture,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院东海水产研究所,上海200090 [2]农业农村部水产品质量安全风险评估实验室(上海),上海200090 [3]上海海洋大学农业部鱼类营养与环境生态研究中心,上海201306 [4]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《食品安全质量检测学报》2020年第22期8235-8239,共5页Journal of Food Safety and Quality

基　　金：中央级公益性科研院所基本科研业务费专项项目(2015M05)。

摘　　要：目的建立间接竞争酶联免疫法(indirect competitive enzyme linkedimmunesorbent assay,IC-ELISA)快速检测鱼体中亚甲基蓝的分析方法。方法以牛血清白蛋白(bovine serum albumin,BSA)为载体蛋白,以天青C(Azure C)为半抗原,采用戊二醛法合成免疫原Azure C-BSA,免疫新西兰大白兔,制备多克隆抗体。通过对包被浓度、抗体浓度和二抗浓度等一系列实验条件的优化,建立检测鱼体中亚甲基蓝的IC-ELISA法。结果获得的多克隆抗体特异性强、灵敏度高,在5~400 ng/mL范围内与抑制率之间线性良好,线性回归方程为Y=0.3658X–0.1867(r2=0.9836),半抑制浓度(50%inhibition concentration,IC50)为75.4 ng/mL,检测限为8.3 ng/mL,样品添加回收率为77.2%~79.3%,相对标准偏差(relative standard deviation,RSD)为5.3%~8.1%。结论该方法灵敏度高、特异性强,可用于鱼体中亚甲基蓝的快速检测。Objective To develop a method for the determination of methylene blue in fish by indirect competitive enzyme linked immunosorbent assay(IC-ELISA).Methods Using bovine serum albumin(BSA)as a carrier protein,Azure C as a hapten,the immunogen Azure C-BSA by glutaraldehyde method was synthesized,then the New Zealand white rabbits were immunized to prepare polyclonal antibody.IC-ELISA method was developed for the determination of methylene blue in fish by optimizing the coating concentration,antibody concentration and secondary antibody concentration.Results The polyclonal antibody was highly specific and sensitive.The linear regression equation was Y=0.3658 X–0.1867(r2=0.9836),with a good linearity with inhibition rate in the range of 5–400 ng/mL.The 50%inhibition concentration(IC50)value and detection limit were 75.4 and 8.3 ng/mL,respectively.The recovery of sample addition was 77.2%–79.3%,with relative standard deviation(RSD)of 5.3%–8.1%.Conclusion This method has high sensitivity and specificity,which could be used for the rapid detection of methylene blue in fish.

关 键 词：亚甲基蓝 多克隆抗体 酶联免疫法 鱼 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]