双抗体夹心酶联免疫吸附法检测鱼糜制品中鸡蛋卵白蛋白

Determination of egg ovalbumin in surimi products by double antibody sandwich enzyme-linked immunosorbent assay

中文关键词:  鸡蛋  卵白蛋白  双抗体夹心酶联免疫吸附法  鱼糜制品

英文关键词:eggs  ovalbumin  sandwich enzyme linked immunosorbent assay  surimi products

中文摘要:

      目的 建立双抗体夹心酶联免疫吸附(sandwich enzyme linked immunosorbent assay, sELISA)法检测鸡蛋卵白蛋白(ovalbumin, OVA)的方法, 组装定量检测鱼糜制品中OVA含量的ELISA检测试剂盒。方法 确定各步骤反应时间, 使用棋盘法确定捕获抗体和检测抗体的最佳工作浓度, 建立检测方法并对其性能进行评价。结果 反应最佳条件为: 抗原孵育20 min, 检测抗体孵育20 min, 酶促反应显色10 min。兔抗OVA抗体为捕获抗体, 工作质量浓度为1 μg/mL, 辣根过氧化物酶(horseradish peroxidase, HRP)标记兔抗OVA抗体为检测抗体, 1:5000 (V:V)稀释。采用四参数逻辑曲线拟合标准曲线, 所建立sELISA方法的检测范围为8.5~200.0 ng/mL, 检出限为3.5 ng/mL, 定量限为8.5 ng/mL。批次内和批次间的变异系数分别为2.31%~4.58%和6.06%~12.23%; 鱼糜基质中的回收率为80%~130%; 测得28种常见食物样品中4种样品的交叉反应率小于0.002%; 试剂盒在4℃保存6个月期间, 标准品检测结果的变异系数为4.29%~18.91%。结论 建立的方法快速、灵敏、准确、稳定性好, 可用于鱼糜制品中鸡蛋OVA的定量检测。

英文摘要:

      Objective To establish a method for the detection of egg ovalbumin (OVA) by double-antibody sandwich enzyme linked immunosorbent assay (sELISA), assemble an ELISA kit for quantitative detection of OVA in surimi products. Methods The reaction time of each step was determined. The optimal working concentration of capture antibody and detection antibody was determined by chessboard method. The detection method was established and its performance was evaluated. Results The optimal reaction conditions were: Incubation of antigen for 20 min, incubation of detected antibody for 20 min, and chromogenic reaction for 10 min. Rabbit anti-OVA antibody was capture antibody, working mass concentration was 1 μg/mL, horseradish peroxidase (HRP) labeled rabbit anti-OVA antibody was detection antibody with a dilution of 1:5000 (V:V). Four parameter logistic curve fitting was used to construct standard curve, the detection range of the established sELISA method was 8.5?200.0 ng/mL, the limit of detection was 3.5 ng/mL, and the limit of quantitation was 8.5 ng/mL. The coefficients of variation within and between batches were 2.31%?4.58% and 6.06%?12.23%, respectively. The recovery rates in surimi matrix were 80%?130%. The cross-reactivity rates of 4 of 28 common food samples were less than 0.002%. The coefficients of variation of the standard test results were 4.29%?18.91% during the 6 months of storage at 4℃. Conclusion The established method is rapid, sensitive, accurate and stable, and can be used for the quantitative detection of egg OVA in surimi products.