基于己烯雌酚-生物素双功能配体的己烯雌酚化学发光酶联免疫吸附检测技术研究

Study on chemiluminescent enzyme-linked immunosorbent assay technology of diethylstilbestrol based on diethylstilbestrol-biotin bifunctional ligand

中文关键词:  双功能配体  生物素-亲和素化学发光酶联免疫吸附试验  己烯雌酚检测  化学发光免疫分析

英文关键词:bifunctional ligand  biotin-avidin-amplified chemiluminescent immunosorbent assay  diethylstilbestrol detection  chemiluminescence immunoassay

中文摘要:

      目的 基于己烯雌酚-生物素双功能配体和生物素-亲和素信号放大系统建立生物素-亲和素化学发光酶联免疫吸附试验(biotin-avidin-amplified chemiluminescent immunosorbent assay, BA-CLIA)法检测牛奶中的己烯雌酚的方法。方法 采用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐/N-羟基琥珀酰亚胺法[N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide, EDC/NHS]制备己烯雌酚-生物素双功能配体; 棋盘滴定法优化己烯雌酚-生物素双功能配体与抗己烯雌酚单克隆抗体的最佳稀释比例; 对有机溶剂、离子强度、pH等影响因素进行优化后建立基于直接竞争化学发光免疫吸附试验检测己烯雌酚的标准曲线; 通过特异性试验测定该方法与己烯雌酚结构类似物的交叉反应率; 最后选取牛奶进行加标回收试验验证该方法在实际样品中检测的可行性。结果 BA-CLIA法对己烯雌酚的半数抑制浓度为3.45 ng/mL, 检出限为0.020 ng/mL, 与传统竞争酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)方法比较, 检测灵敏度提高了6.22倍; BA-CLIA法与己烯雌酚类似物(双烯雌酚、雌二醇、雌三醇和双酚A)的交叉反应性较低, 显示了较好的特异性; 采用该方法检测牛奶中的己烯雌酚, 平均加标回收率为92.80%~95.74%, 变异系数小于等于8.05%。结论 该方法灵敏度高、特异性好, 适用于牛奶中的己烯雌酚的测定。

英文摘要:

      Objective To establish a method of biotin-avidin-amplified chemiluminescent immunosorbent assay (BA-CLIA) for the detection of diethylstilbestrol in milk based on diethylstilbestrol-biotin bifunctional ligand and biotin-avidin signal amplification system. Methods N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride/ N-hydroxysuccinimide, (EDC/NHS) was used to prepare the diethylstilbestrol-biotin bifunctional ligand. The optimal dilution ratio of diethylstilbestrol-biotin bifocal ligand and monoclonal antibody against diethylstilbestrol was optimized by checkerboard titration. A standard curve for the detection of diethylstilbestrol based on direct competitive chemiluminescence immunosorbent assay was established after optimizing the influencing factors such as organic solvent, ionic strength, pH, etc. The cross-reaction rate of this method with diethylstilbestrol structural analogs was determined by specificity test. Finally, milk was selected for spiked recovery test to verify the feasibility of this method in actual samples. Results The median inhibitory concentration of BA-CLIA method for diethylstilbestrol was 3.45 ng/mL, and the limit of detection was 0.020 ng/mL. Compared with the traditional competitive enzyme linked immunosorbent assay (ELISA) method, the detection sensitivity was increased by 6.22 times. The BA-CLIA method had low cross-reactivity with diethylstilbestrol analogs (diethylstilbestrol, estradiol, estriol, and bisphenol A), showing better specificity. Using this method to detect diethylstilbestrol in milk, the average spiked recoveries of standard addition were 92.80%-95.74%, and the coefficient of variation was less than or equal to 8.05%. Conclusion The method has high sensitivity and specificity and is suitable for the determination of diethylstilbestrol in milk.