牛乳过敏原β-乳球蛋白间接竞争ELISA检测方法的建立

A method for detecting β-lactoglobulin in cow milk allergens by indirect competitive enzyme-linked immunosorbent assay

检测牛乳中的主要过敏原β-乳球蛋白,利用β-乳球蛋白纯品免疫新西兰白兔,制得兔抗牛β-乳球蛋白的多克隆抗体,以β-乳球蛋白包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG为二抗、四甲基联苯胺（TMB）为底物,建立了牛乳中β-乳球蛋白的间接竞争酶联免疫检测法（ELISA）。确定了包被抗原质量浓度为0.5μg/mL,一抗最佳稀释度为1∶6 400,酶标二抗稀释度为1∶10 000。结果达到批内误差3.87%,批间误差6.51%;检测的线性范围为0.05～1.00μg/mL,标准曲线回归方程y=-1.930 2x-0.124 1,相关系数为0.994 5,说明所建立的方法灵敏度高,具有良好的准确性和重复性。

β-lactoglobulin is the main allergen in cow milk and to detect it in the milk, an indirect competitive enzymelinked immunosorbent assay （ELISA） was developed. A polyclonal antibody was prepared by immunizing New Zealand rabbits with β-lactoglobulin. Results showed that the optimal concentrations of the coating antigen, polyclonal antibody against β-lactoglobulin and horseradish peroxidase conjugated goat anti-rabbit IgG were 0.5 μg/mL, 1 ： 6 400 and 1 ： 10 000 respectively; The coefficients of variation of intra-and inter-assays were 3.87% and 6.51%. The standard curve of indirect competitive ELISA was also obtained with the regression equation as y = - 1. 930 2x - 0. 124 1 （ r^2 = 0.994 5） and the curve was linear within the range of 0.05- 1.00 μg/mL. Thus, this method has good sensitivity for the detection of β-lactoglobulin in cow milk.