燕窝唾液酸糖蛋白间接竞争酶联免疫检测方法的建立

Establishment of indirect competitive enzyme linked immunosorbent assay for detection of sialoglycoprotein of edible bird’s nest

中文关键词:  燕窝  唾液酸糖蛋白  间接竞争酶联免疫分析法

英文关键词:edible bird’s nest  sialoglycoprotein  indirect competitive enzyme-linked immunosorbent assay

中文摘要:

      目的 建立间接竞争酶联免疫(indirect competitive enzyme linked immunesorbent assay, IC-ELISA)检测燕窝中唾液酸糖蛋白的分析方法。方法 以唾液酸糖蛋白为抗原, 制备单克隆抗体, 对IC-ELISA步骤中相关参数进行优化, 确定最佳反应条件。结果 最佳包被抗原为1:2000, 抗体稀释倍数为1:5000; 封闭液浓度为1%牛血清白蛋白(bovine serum albumin, BSA), 封闭时间为150 min; 竞争时间30 min; 酶标二抗浓度稀释倍数为1:1000, 孵育时间30 min; 显色时间为10 min。建立的IC-ELISA检测方法半抑制浓度(50% inhibition concentration, IC50)为8.61 μg/mL, 线性范围为2.23~33.25 μg/mL, 回收率为82.7%~92.0%, 相对标准偏差(relative standard deviation, RSDs)为1.7%~8.9%。结论 该方法灵敏度较高、特异性强, 可以有效地鉴别假冒伪劣燕窝产品, 具有较好的实际应用价值。

英文摘要:

      Objective To establish an indirect competitive enzyme linked immunosorbent assay (IC-ELISA) method for the detection of sialoglycoprotein in edible bird’s nest. Methods Monoclonal antibody was prepared with sialoglycoprotein as antigen, and the relevant parameters in IC-ELISA step were optimized to determine the optimal reaction conditions. Results The optimal coating antigen was determined to be 1:2000, and the dilution of antibody was 1:5000. The concentration of blocking liquid was 1% bovine serum albumin (BSA) and the blocking time was 150 min. Competition time was 30 min. The concentration dilution of enzyme-conjugate secondary antibody was 1:1000. The incubation time was 30 min and colouration time was 10 min. The results showed that the 50% inhibition concentration (IC50) of the established IC-ELISA was 8.61 μg/mL. The linear range was 2.23?33.25 μg/mL and the recoveries were between 82.7%?92.0%, with relative standard deviations (RSDs) of 1.7%–8.9%. Conclusion This method has high sensitivity and specificity, which can be used for effectively identify fake bird’s nest products and has good practical application value.