定量检测牛奶中蛋白质ELISA方法的建立

本试验旨在建立特异检测牛奶蛋白质含量的ELISA方法。利用提纯酪蛋白免疫试验动物,获得针对酪蛋白的多克隆抗体;利用棋盘格试验确定抗原抗体的最佳作用浓度,并优化各步骤反应条件;根据标准样品建立牛奶中蛋白质含量的定量ELISA检测方法,并对该方法的特异性、敏感性、检测范围及重复性进行评价。通过试验确定封闭液为1% BSA,37 ℃孵育120 min;血清抗体工作浓度为1∶12000稀释,37 ℃孵育90 min;酶标二抗最适浓度为1∶3000稀释,37 ℃孵育30 min。牛奶酪蛋白检出范围为0.063~0.500 μg/mL;该方法具有良好的特异性、敏感性、重复性,回归率范围为97.6%~123%。成功建立了牛奶蛋白含量的定量ELISA检测方法。

Development of an Optimized ELISA for the Quantitative Detection of Protein in the Milk

This experiment is aimed to develop a ELISA method to detect the precise content of protein in the milk specificly. Immune the rabbit with pure casein,the polyclonal antibodies against casein were obtained. The best working concentration of antigen and antibody was determined using the checkerboard test, and the reaction conditions of each steps were optimized. The 1% BSA was served as blocking agent, and its incubation time was 120 min. The optimal dilution and incubation time of serum sample and enzyme-labeled second antibody were 1∶12000 for 90 min and 1∶3000 for 30 min respectively. The detection range of casein was 0.063 to 0.500 μg/mL. The method had good specificity, accuracy, repeatability, the range of regression rate was 97.6% and 123%.The ELISA method for detecting the protein content of milk was successfully established.