扇贝过敏原荧光免疫层析检测方法的构建与应用

Construction and application of a fluorescence immunochromatographic assay for allergen detection of scallops

中文关键词:  时间分辨荧光微球  免疫层析  扇贝过敏原  原肌球蛋白  快速检测

英文关键词:time-resolved fluorescent microspheres  immunochromatography  scallop allergens  tropomyosin  rapid detection

中文摘要:

      目的 构建一种简便、快速、可降低非特异性荧光干扰的时间分辨免疫层析检测方法, 实现快速检测扇贝中原肌球蛋白(tropomyosin, TM)过敏原。方法 本研究采用双抗体夹心免疫层析法, 以含有铕(Eu)纳米微粒的荧光微球作为标签偶联兔抗TM多克隆抗体, 制备荧光探针并对其进行表征。以4 mg/mL兔多克隆抗体作为T线, 羊抗兔免疫球蛋白G (immunoglobulin g, IgG)作为C线组装免疫层析试纸条。结果 本研究组建的试纸条视觉检出限为0.05 μg/mL, 仪器检出限为0.01 μg/mL。试纸条除对虾蟹有交叉反应外, 对其他10余种物种无明显交叉反应。加标样品批内和批间变异系数分别为3.71%~7.94%和12.09%~12.80%, 在不含TM的4种食物基质中加入浓度由低到高的TM, 检测结果与实际加标情况相符。结论 本检测方法准确性良好、灵敏度高、特异性强, 可在多种食物基质中实现对扇贝TM的快速检测。

英文摘要:

      Objective To establish a simple, rapid and time-resolved immunochromatography assay which can reduce non-specific fluorescence interference, and realize the rapid detection of tropomyosin (TM) allergens in scallops. Methods In this study, double antibody sandwich immunochromatography was used to couple rabbit anti-TM polyclonal antibody with fluorescent microspheres containing europium (Eu) nanoparticles as a label, and the fluorescent probes were prepared and characterized. The immunochromatographic test strip was assembled with 4 mg/mL rabbit polyclonal antibody as the T-line and goat anti-rabbit immunoglobulin g (IgG) as the C-line. Results The visual limit of detection of the test strip constructed in this study was 0.05 μg/mL, and the limit of detection of the instrument was 0.01 μg/mL. Except for cross reaction with shrimp and crab, the test strips showed no significant cross reaction with more than other 10 species. The coefficient of variation within and between batches of the spiked samples were 3.71%?7.94% and 12.09%?12.80% respectively. TM was added into the 4 kinds of food matrices that did not contain TM from low concentration to high concentration, and the test results were consistent with the actual spiked conditions. Conclusion This method has good accuracy, high sensitivity and strong specificity, and can be used for rapid detection of scallop TM in various food substrates.