为了建立快速、敏感、特异的雌二醇(E2)残留免疫检测方法,本研究应用胶体金免疫层析技术,研究制备一种快速检测牛奶中E2的方法.采用柠檬酸三钠还原法制备胶体金颗粒,采用物理吸附法将E2兔多克隆抗体偶联至胶体金,经过优化抗原包被量和多克隆抗体标记量等反应条件组装成检测试纸条,测定检测试纸的灵敏度、特异性、重复性和加速保存等参数,并对E2类似物交叉反应和牛奶基质干扰反应进行了测定.结果表明,试纸条最优包被抗原浓度为2 mg/mL,抗体浓度为20μg/mL.采用消线法判定结果,检测时间10 min,PBS缓冲液中E2检测限为10 μg/L,该方法与雌三醇的交叉反应率为40％,与戊酸雌二醇、苯甲酸雌二醇、雌酮、己烯雌酚、炔雌醚、乙炔雌二醇、壬基酚等类似物均无可见交叉反应,牛奶样品经2倍稀释消除基质干扰直接用于检测,该方法在牛奶样品中的判定值为20 μg/L.本研究建立的E2胶体金试纸条使用简单方便,适合现场快速检测牛奶中E2残留.%In order to develop a sensitive and specific method for the detection of estradiol residues (E2) in milk,a colloidal gold immunochromatographic method was established.Colloidal gold particles were prepared by trisodium citrate reduction method and labeled with rabbit polyclonal antibodies (PcAb) by physical adsorption method.After optimization of reaction conditions such as the amount of coating antigen and labeled antibody to assemble test strip,the sensitivity,specificity,repeatability and accelerated preservation of the test strip were determined.The estrogen analogue cross reaction and milk matrix interference reaction were also measured.The results showed that the optimized concentration of E2-OVA antigen was 2 mg/mL and the optimized antibody concentration was 20 μg/mL,visual detection limits of E2 was 10 μg/L in PBS within 10 min.The cross reaction rate of this method with estriol was 40 ％,there were no negligible crossreactivities with other estrogen compounds including estradiol valerate,estradiol benzoate,estrone,diethylstilbestrol,quinestrol,ethinyloestradiol and nonylphenol.Milk samples only needed two times diluted before analysis,and the results could judge by naked eye after 10 min with cut-off of 20 μg/L.The results demonstrated that the developed method was suitable for rapid on-site screening of E2 residues in milk samples.
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