荧光微球免疫层析法定量检测牛乳中酪蛋白

Fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk

中文关键词:  酪蛋白  免疫层析  荧光微球  定量检测

英文关键词:casein  lateral flow assay  fluorescent microspheres  quantitative detection

中文摘要:

      目的 研究用荧光微球免疫层析法定量检测牛乳中的酪蛋白。方法 本文以酪蛋白为抗原, 免疫新西兰大白兔制备抗酪蛋白的多克隆抗体, 将纯化后的多克隆抗体通过EDC介导法与荧光微球进行偶联, 将滤纸、样品垫、结合垫、NC膜和吸水纸组装成试纸条, 用此荧光微球免疫层析法定量检测牛乳中的酪蛋白。结果 试纸条在25 min内就能判定结果, 最低检测限为100 ng/mL, 该方法与BSA、OVA均无交叉反应, 具有很好的特异性。检测酪蛋白浓度为100.0、500.0、1000.0 ng/mL的样品, 试纸条的批内回收率分别为(89.03±5.2)%、(93.47±6.9)%和(91.2±7.8)%, 批间回收率分别为(87.69±6.2)%、(92.73±8.3)%、(89.82±8.5)%。结论 初步建立了一种快速、方便、高灵敏度的荧光微球免疫层析方法用以检测牛乳制品中的过敏原——酪蛋白。

英文摘要:

      Objective To develop a fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Methods Casein was used as the antigen to immunize the New Zealand rabbits to make the corresponding polyclonal antibody against the antigen, and EDC mediated method was used to couple the purified polyclonal antibody with fluorescent microspheres. The filter pad, sample pad, conjugate pad, NC membrane, and absorption pad were assembled to develop the fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Results The results could be obtained within 25 min without sophisticated equipment, and the limit of detection of the test strip was 100 ng/mL. The developed assay was highly specific to the casein protein as a result of that it had no cross-reactivity with other non-target proteins, such as BSA and OVA. The recovery rates of the intra-assay at casein concentrations of 100.0, 500.0, and 1000.0 ng/mL were (89.03±5.2)%, (93.47±6.9)% and (91.2±7.8)%, respectively, while the inter-assay were (87.69±6.2)%, (92.73±8.3)% and (89.82±8.5)%, respectively. Conclusion The fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk with the advantages of rapid, convenient, and high sensitivity was established preliminarily.