Development and characterization of an immunoaffinity column for the detection of danofloxacin residues in milk samples

Abstract In this study, an immunoaffinity column was prepared by covalently coupling of polyclonal antibody against danofloxacin (DAN) with chitosan microparticles and its potential application to selective extraction of DAN from food samples was also evaluated. The produced antibody exhibited high sensitivity, with an IC50 value of 4.56 ng/mL towards DAN, and good specificity, with less than 0.5% of cross-reactivity towards DAN structurally related compounds. The developed immunoaffinity column had an approximate binding capacity of 2300 ng/mL gel and a relative standard deviation (RSD) of 3.7% among columns. The immunoaffinity columns were then challenged with DAN-fortified milk samples, recoveries of DAN were found to be in the range of 83.8% to 98.8%, indicating the prepared immunoaffinity column is suitable for use as an efficient extraction method to detect DAN residues in food products.摘要 本研究将针对达氟沙星（danofloxacin, DAN）的多克隆抗体与壳聚糖微球进行共价偶联，制备得到免疫亲和柱（immunoaffinity column），并评估其用于从食品样品中选择性萃取达氟沙星的应用潜力。所制备的抗体展现出优异的灵敏度，对达氟沙星的半抑制浓度（IC50）为4.56 ng/mL，同时具备良好的特异性，对达氟沙星结构类似物的交叉反应率低于0.5%。所开发的免疫亲和柱的结合容量约为2300 ng/mL凝胶，柱间相对标准偏差（relative standard deviation, RSD）为3.7%。随后采用达氟沙星加标牛奶样品对该免疫亲和柱进行性能验证，结果显示达氟沙星的回收率介于83.8%至98.8%之间，表明所制备的免疫亲和柱可作为高效萃取手段，用于检测食品中的达氟沙星残留。