生物素-亲和素放大酶联免疫吸附法测定牛奶中雌三醇  

Biotin-avidin Mediated Competitive Enzyme-linked Immunosorbent Assay for Determination of Estriol in Milk

作　　者：梁祖培 冯观萍 吴民富 孙魁魁 张燕 刘辉 Liang Zupei;Feng Guanping;Wu Minfu;Sun Kuikui;Zhang Yan;Liu Hui(Guangdong Testing Institute of Product Quality Supervision, Foshan 528300;TransGen B iotech, Beijing 100192;Department of Food Science, Foshan Polytechnic, Foshan 528137, China)

机构地区：[1]广东产品质量监督检验研究院,广东佛山528300 [2]北京全式金生物技术有限公司,北京100192 [3]佛山职业技术学院食品科学系,广东佛山528137

出　　处：《广东化工》2018年第11期72-75,共4页Guangdong Chemical Industry

基　　金：广东省省级科技计划项目(2013B090600059);2017年佛山市自筹经费类科技计划项目(2017AB004141)

摘　　要：建立了检测牛奶中雌三醇的生物素-亲和素放大酶联免疫吸附测定法(BA-ELISA),实验最佳工作条件为,包被原浓度为125 ng/m L,生物化抗体的稀释倍数为1∶16000,缓冲体系为pH=7.4的0.01 mol/L的PBS缓冲溶液,体系中甲醇浓度低于5%,生物素化抗体孵育时间和亲和素-HRP孵育时间均为30 min。在此最佳条件下,方法的检测范围(IC20~IC80)为0.149~3.27 ng/m L,灵敏度IC50=0.689 ng/m L。建立了液-液提取样品前处理方法,前处理净化后稀释10倍可消除基质效应,雌三醇牛奶样品加标回收率为94.9%~115.2%,CV<15%。与ELISA法相比,BA-ELISA具有更高的灵敏度,适于低浓度雌三醇的检测。A rapid and sensitive method based on biotin-avidin mediated competitive enzyme-linked immunosorbent assay（BA-ELISA） was established for the determination of estriol in milk. Through the optimization experiment, the coating concentration was 125 ng/mL, the biotinylated antibody dilution factor was 1： 16000. The effect of 0.01 mol/L phosphate buffer solution, pH=7.4, 5 % methanol and 30 min competitive reaction time was determined as the biotin-avidin enzyme linked immumosobent assay（BA-ELISA） for the interaction of estriol and antibody in BA-ELISA. And under the optimum conditions, The IC50=0.689 ng/mL, the detection range（IC20-IC80） was 0.149-3.27 ng/mL. The samples are pretreated by means of the liquid-liquid extraction and diluted 15 times to eliminate the matrix effect. The recoveries of estriol spiked in milk were from 94.9 % to 115.2 %, CV was less than 15 %. Comparing the result of traditional ELISA, the present BA-ELISA method had much higher sensitivity for estriol.The experimental results indicated that the present BA-ELISA method can be used to quickly monitor the estriol in a simple and fast.

关 键 词：雌三醇 单克隆抗体 生物素-亲和素放大酶联免疫吸附法 液-液提取法 

分 类 号：O647.3[理学—物理化学] TS252.7[理学—化学]