榛子过敏原双抗夹心酶联免疫吸附方法的建立及应用

Establishment and application of sandwich enzyme-linked immunosorbent assay for hazelnut allergen

中文关键词:  榛子  过敏原  酶联免疫吸附方法  检测

英文关键词:hazelnut  allergen  enzyme linked immunosorbent assay  detection

中文摘要:

      目的 建立榛子过敏原双抗夹心酶联免疫吸附法(sandwich enzyme-linked immunosorbent assay, sELISA)快速检测食品中微量榛子的方法。方法 对两株抗体进行配对, 确定捕获抗体和检测抗体, 利用棋盘法确定捕获抗体、检测抗体、辣根过氧化物酶标记的兔抗大鼠IgG(horseradish peroxidase-IgG, HRP-IgG)的最佳稀释浓度, 然后对各工作步骤的孵育时间进行优化, 并应用于实际食品的检测。结果 使用兔抗作为捕获抗体, 鼠抗作为检测抗体, 兔抗血清稀释度为1:10000 (V:V)、鼠抗血清稀释度为1:10000 (V:V)、HRP-兔抗大鼠稀释度为1:20000 (V:V)。各步骤最佳反应时间为抗原孵育30 min、检测抗体孵育30 min、酶标二抗孵育45 min及四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine, TMB)显色8 min。此方法的线性回归方程为Y=0.4504 ln(X)+2.1194, r2=0.9934, 定量限为10 ng/mL、检出限为0.1 ng/mL。该方法特异性强, 仅与核桃产生轻微的交叉反应, 在面包、酸奶和咖啡基质中的回收率在80%到120%之间, 批内、批间变异系数均小于15%。结论 本方法具有较高的灵敏度和特异性, 适用于商业加工食品中榛子残留的快速、准确检测。

英文摘要:

      Objective To establish a method for rapid determination for trace hazelnuts in food by hazelnut allergen sandwich-enzyme linked immunosorbent assay (sELISA). Methods The two antisera were paired to determine the captured antibody and detection antibody, the optimal dilution concentration of capture antibody, detection antibody and horseradish peroxidase-labeled rabbit anti-rat IgG (HRP-IgG) were determined by chessboard method, then the incubation time of each working step were optimized and applied it to the actual food detection. Results The rabbit antibody was used as the capture antibody, and the mouse antibody was used as the detection antibody. The dilution of rabbit antiserum was 1:10000 (V:V), mouse antiserum was 1:10000 (V:V), and HRP-rabbit antirat was 1:20000 (V:V). The optimal reaction time of each step was determined as 30 min of antigen incubation, 30 min of detection antibody incubation, 45 min of HRP-rabbit anti-mice incubation and 8 min of 3,3’,5,5’-tetramethylbenzidine (TMB) color rendering. The linear regression equation of this method was Y=0.4504 ln(X)+2.1194, r2=0.9934, the limit of quantitation was 10 ng/mL, the limit of detection was 0.1 ng/mL. The method had strong specificity and only slightly cross-reacted with walnuts. The recovery rates in bread, yogurt and coffee matrix were between 80% and 120%, and the intra-assay and inter-assay coefficients of variation were both less than 15%. Conclusion This method has high sensitivity and specificity, and is suitable for rapid and accurate determination of hazelnut residues in commercial processed foods.