大豆过敏原夹心ELISA和竞争ELISA方法的建立及其在加工食品中应用研究

本文以大豆混合过敏原为目标，建立了快速、便捷检测大豆过敏原的夹心酶联免疫吸附方法（sandwich-enzyme linked immunosorbent assay）和间接竞争酶联免疫吸附方法（indirect competitive enzyme-linked immunosorbent assay），通过实际加工样品的回收实验、加标食品回收实验以及对真实食物样本的检测，对这两种方法进行了比较，确定了各自的适用范围。结果表明，夹心ELISA方法标准品浓度在0.0078~30 μg/mL范围内呈现出良好的线性关系，曲线方程为y=0.2333x+0.0692，决定系数R2=0.995。竞争ELISA方法的检测范围为10~100000 ng/mL，最低检测限为10 ng/mL。对购入橙汁进行加标回收实验，夹心ELISA检测后的回收率要高于竞争ELISA检测后的回收率，达100%以上；而对成分和加工方式都比较复杂的巧克力、牛肉酱、面包或蛋糕来说，竞争ELISA检测后的回收率要高于夹心ELISA检测后的回收率。对发酵类食物进行检测，竞争ELISA方法检测到的浓度要高于夹心ELISA，而对成分比较简单的食物比如芝麻糊、豆奶等进行检测时，夹心ELISA的检测浓度要略高于竞争ELISA。综上，竞争ELISA方法更适用于食物基质复杂，经过深度加工的食品，而夹心ELISA方法更适用于食物成分简单，轻加工后的食品，两种方法在各自的适用范围内均能实现较准确的检测。

Development of Sandwich and Competitive ELISA Formats to Determine Soybean Allergen: Evaluation of Their Performance to Detect Soy in Processed Food

Taking soybean mixed allergens as the target, a sandwich-enzyme linked immunosorbent assay and an indirect competitive enzyme-linked immunosorbent assay for the rapid and convenient detection of soybean allergens were established. The two methods were compared through the recovery experiment of actual processed samples, the recovery experiment of spiked food and the detection of real food samples, and their respective application scopes were determined. Sandwich ELISA method, the standard concentration was in the range of 0.0078~30 μg/mL, showing a good linear relationship, the curve equation was y=0.2333x+0.0692 with coefficient of determination R2=0.995. The detection range of the competitive ELISA method was 10~100000 ng/mL, and the lowest detection limit was 10 ng/mL. The recovery rate of orange juice after sandwich ELISA was higher than that after competitive ELISA, reaching more than 100%. For chocolate, beef sauce, bread and cake with complex ingredients and processing methods, the recovery rate after competitive ELISA was higher than that after double-antibody sandwich ELISA. In the detection of soybean allergens in fermented foods, the concentration detected by competitive ELISA was higher than that detected by sandwich ELISA, while in the detection of simple ingredients such as sesame paste and soy milk, the concentration detected by sandwich ELISA was slightly higher than that detected by competitive ELISA. In general, the competitive ELISA method was more suitable for the food with complex food matrix and deeply processed, while the sandwich ELISA method was more suitable for the food with simple food ingredients and lightly processed food, both of the two methods can achieve more accurate detection in their respective application scope.