动物过敏原牛血清白蛋白间接竞争ELISA检测方法的建立

[目的] 制备鼠抗牛血清白蛋白(bovine serum albumin,BSA)单克隆抗体,在此基础上建立可用于定量检测动物过敏原BSA含量的间接竞争酶联免疫检测方法(IC-ELISA)。[方法] 采用BSA纯品为抗原免疫BALB/c小鼠,通过杂交瘤细胞融合技术制备抗BSA单克隆抗体,并对其特异性进行鉴定;在此基础上,建立BSA间接竞争ELISA检测方法,并对其分析条件进行优化。[结果] 采用自行制备的特异性抗BSA单克隆抗体,建立间接竞争ELISA检测方法,优化得到最佳反应条件:抗原最佳包被浓度为0.25 μg？mL-1,单抗最佳工作浓度为1:32 000,酶标二抗最佳工作浓度为1:10 000,最低检测限为6.71 ng？mL-1,线性检测范围为3.593 8~460 ng？mL-1。线性回归直线方程为y=-47.448 9x+135.866 3,相关系数R2=0.997 3,P<0.000 1。[结论] 该方法特异性强、灵敏度高、可靠性好,适用于食品中过敏原BSA的现场快速检测。
[Objectives] An indirect competitive enzyme-linked immunosorbent assay(indirect competitive enzyme-linked immunosorbent assay,IC-ELISA)was developed for the quantitative detection of bovine serum albumin(BSA)using a self-prepared mouse anti-BSA monoclonal antibody. [Methods] BSA standard protein was immunized to BALB/c mice to prepare the specific monoclonal antibody against BSA by hybridoma technology. An IC-ELISA method for BSA based on monoclonal antibodies was developed,and its analysis conditions were optimized. [Results] The IC-ELISA method was established using specific monoclonal antibodies. The optimal concentrations of the coating antigen,monoclonal antibody against BSA and HRP conjugated goat anti-mouse IgG were 0.25 μg？mL-1,1:32 000 and 1:10 000,respectively. The limited detection was 6.71 ng？mL-1 in a linear range of 3.593 8-460 ng？mL-1. The regression equation of standard curve was y=-47.448 9x+135.866 3(the correlation coefficient R2=0.997 3,P<0.000 1). [Conclusions] The IC-ELISA method constructed in the paper has high specificity,good sensitivity and reliability,and can be used for rapid detection of animal allergen BSA in food