High throughput detection of antibiotic residues in milk by time-resolved fluorescence immunochromatography based on QR code

ABSTRACT

Herein, we have successfully established a novel, rapid, and simple lateral-flow immunoassay based on time-resolved fluorescence and biotin-streptavidin to detect the residues of various antibiotics in milk. The fluorescence signal and sensitivity of immunochromatography were enhanced through biotinylated antibody coupled with streptavidin europium microspheres. Moreover, due to the use of a QR Code and fluorescent reader, quantitative detection and real-time data uploading can be achieved. Under the optimal conditions, the various antibiotic residues were detected in the milk samples. The results showed that the limits of detection of tylosin, lincomycin and doxycycline were 0.10, 0.06, and 0.27 ng/mL, respectively. The recoveries of the spiked milk samples were 88.9%~127%, with coefficients of variation less than 11%, and the test strip can be stored at room temperature for 12 months. This study shows that the proposed time-resolved fluorescence immunoassay is sensitive, rapid and reliable, and has the potential to be used for detection of veterinary antibiotic residues in food safety fields.     

牦牛肉中阿维菌素残留的时间分辨荧光免疫层析检测方法的建立

为建立定量检测牦牛肉中阿维菌素残留的时间分辨荧光免疫层析方法,以羧基化铕微球作为荧光标记物,与阿维菌素鼠单克隆抗体G10703进行共价偶联后喷涂于释放垫上,将阿维菌素抗原和羊抗鼠免疫球蛋白G分别包被于硝酸纤维素膜上作为检测线（T）和质控线（C）,制备免疫层析试纸条;利用牦牛肉样本阿维菌素添加量和对应的T线与C线荧光信号峰值比值（T/C）拟合得到四参数标准曲线,建立牦牛肉中阿维菌素残留的时间分辨荧光免疫层析检测方法,并对所建立方法的灵敏度、准确度、特异性、精密度和稳定性等进行评价,用液相色谱-串联质谱方法对所建立方法的性能进行确证。结果表明：本方法对牦牛肉中阿维菌素的定量限为1.0 μg/kg,加标回收率为86.9%～105.2%,与阿维菌素、伊维菌素、多拉菌素及依普菌素的交叉反应率分别为100.0%、73.1%、42.6%和97.5%,批内变异系数为5.4%～9.5%,批间变异系数为7.5%～11.2%,该方法准确度、精密度、灵敏度均较高,且性能稳定。     

蘑菇中鹅膏毒肽间接竞争ELISA检测方法的建立

目的:旨在建立一种检测蘑菇中鹅膏毒肽（Amanitin, AMA）的间接竞争酶联免疫吸附方法（Indirect competitive enzyme-linked immunosorbent assay, ic-ELISA）。方法:通过EDC/NHS在α-鹅膏毒肽的羧基位置引入6-氨基己酸得到半抗原,并进一步与不同的载体蛋白偶联制备免疫原和包被原。之后,利用免疫原免疫Balb/c小鼠制备单克隆抗体。基于所获得的抗体,并通过对包被原和抗体工作浓度、包被条件、封闭条件、酶标二抗工作浓度和孵育时间等条件的优化,建立了蘑菇中鹅膏毒肽间接竞争ELISA检测方法,最后对所建立方法的灵敏度、添加回收率、批内及批间变异等参数进行了评价。结果:合成的半抗原分子量为1033.12,经MALDI-TOF鉴定免疫原的偶联比约为10.03。基于杂交瘤技术制备、筛选出鼠单克隆抗体13H4的IC₅₀为1.91 μg/L。基于所获得单克隆抗体,所建立蘑菇中鹅膏毒肽间接竞争ELISA方法的检出限为0.88 μg/kg,添加回收率为85.66%~113.05%,批内变异系数为5.35%~9.54%,批间变异系数小于15%。结论:本研究所建立的ic-ELISA方法准确度、精密度、灵敏度较高、性能稳定,为突发毒蘑菇中毒事件的毒原分析提供了一种简便、可靠的快速检测方法。     

鸡、鸭肉中金刚烷胺、金刚乙胺、索金刚胺间接竞争ELISA检测方法研究

目的:建立一种检测鸡肉、鸭肉中金刚烷胺、金刚乙胺、索金刚胺的间接竞争酶联免疫吸附方法(Indirect competitive enzyme-linked immunosorbent assay,ic-ELISA).方法:本研究基于间接竞争酶联免疫方法的原理,在酶标板微孔中预包被偶联抗原,样本中含有的金刚烷胺、金刚乙胺...     

时间分辨荧光免疫层析定量检测牦牛肉中喹诺酮类药物

目的:本研究旨在建立一种简单、快速、高灵敏的牦牛肉中喹诺酮类药物(环丙沙星、恩诺沙星、诺氟沙星、氧氟沙星、达氟沙星等)残留的荧光定量免疫层析检测方法。方法:将环丙沙星鼠单克隆抗体G10785与羧基化铕微球进行偶联标记,环丙沙星抗原和羊抗鼠二抗分别包被于硝酸纤维素膜(NC)上作为检测线(T)和控制线(C);根据T线荧光信号值与C线荧光信号值的比值(T/C)和样本中喹诺酮类药物浓度建立定量标准曲线;对所建立方法的灵敏度、准确度、精密度等进行评价,并将其与LC-MS/MS方法进行对比。结果:本研究所建立方法的定量限<0.96 μg/kg,批内回收率为82.94%~111.27%,变异系数为4.13%~9.90%,批间回收率为88.86%~107.05%,变异系数为3.86%~9.39%。与环丙沙星、恩诺沙星、诺氟沙星、氧氟沙星、达氟沙星的交叉反应率分别为100%、77.36%、107.66%、93.55%和50.92%。结论:本研究所建立的时间分辨荧光免疫层析检测方法准确度、精密度、灵敏度较高,性能稳定,具有良好的应用前景。