基于定向标记抗体的虾原肌球蛋白量子点免疫层析方法的建立及应用

Establishment and application of quantum dots immunochromatographic method based on directed labeling antibody to detection shrimp tropomyosin

中文关键词:  定向标记  量子点  原肌球蛋白  免疫层析试纸条  快速检测

英文关键词:directed labeling  quantum dots  tropomyosin  immunochromatographic test strips  rapid detection

中文摘要:

      目的 利用量子点定向标记的单克隆抗体, 建立竞争荧光免疫层析方法快速检测食品中虾原肌球蛋白(tropomyosin, TM)。方法 使用杂交瘤细胞株免疫Balb/c小鼠, 从小鼠腹水中制备得到单克隆抗体, 将氨基化量子点通过具有抗体Fc端生物亲和性的重组蛋白共价固定于抗体Fc端。以量子点定向标记抗体作为检测抗体, 以南美白对虾中的TM作为检测靶标, 基于竞争法原理组装免疫层析试纸条。在对条件参数进行优化后, 应用于市售食物样品中TM的检测。结果 所建立方法的灵敏度为0.96 μg/mL (%P=50%), 检出限为0.15 μg/mL (%P=20%), 检测结果与文献中报道的相当。试纸条批内差和批间差的变异系数分别为6.81%~8.86%和8.27%~14.67%, 均小于15%, 在可接受范围之内。同时试纸条显示出了良好的特异性与准确性, 实际样品的检测结果与过敏原标签一致。结论 本研究所建立的荧光免疫层析试纸条检测方法适用于TM的快速、高效检测。

英文摘要:

      Objective To develop a competitive fluorescent immunochromatographic method for the rapid detection of shrimp tropomyosin (TM) in food by using quantum dots directed labeling monoclonal antibody. Methods The hybridoma cell line was used to immunize Balb/c mice, and monoclonal antibodies were prepared from mouse ascites. Aminated quantum dots were covalently immobilized at the Fc-terminal of the antibody by the recombinant protein that has bio-affinity for the Fc-terminal of the antibody. The immunochromatographic test strips were assembled based on the principle of competition method using quantum dots directed labeling antibody as the detection antibody and TM in South American white shrimp as the detection target. After the optimization of the condition parameters, it was applied to the detection of TM in commercially available food samples. Results The sensitivity of the established detection method was 0.96 μg/mL (%P=50%) and the limit of detection was 0.15 μg/mL (%P=20%), which was comparable to the results reported in the literature. The intra- and inter-batch coefficients for variation of the test strips were less than 15%, ranging from 6.81%?8.86% and 8.27%?14.67%, respectively, which were within the acceptable range. It also showed good specificity and accuracy, and the results of the actual samples were consistent with the allergen labels. Conclusion The fluorescent immunochromatographic test strip detection method established in this study is suitable for the rapid and efficient detection of TM.