一种基于荧光微球标记的卡那霉素免疫层析定量方法的建立

Development of a Microsphere-Based Fluorescence Immunochromatographic Assay for Detection of Kanamycin

[目的]卡那霉素是一种在奶牛兽医临床常用药物,其在牛奶中的残留不容忽视。荧光微球作为一种新型的荧光示踪物,近年来在小分子快速检测方面呈现出独特的优势,试验开展了卡那霉素荧光微球免疫层析定量方法的研究,旨在为牛奶中卡那霉素药物的快速筛选提供新型有效的检测手段。[方法]采用戊二醛法和过碘酸钠法制备了两种卡那霉素的包被抗原(KANA-GA-OVA1、KANA-I2-OVA2),抗卡那霉素的单克隆抗体通过蛋白G免疫亲和柱进行纯化后,采用碳二亚胺法与荧光微球进行了共价偶联,成功制备了抗体-荧光微球标记物。免疫层析试纸条方法采用微孔测定法。包被卡那霉素包被抗原的NC膜作为固相载体,荧光微球标记的单克隆抗体与样品混合,在微孔膜的毛细管作用下,包被抗原和待测样本中的药物来共同竞争有限的单克隆抗体,通过荧光测定包被抗原结合的抗体量来评价待测样本中卡那霉素药物的含量。[结果]灵敏度试验表明,微孔中加入0、6.25、12.5、25、50、100、200μg·L-17个浓度梯度的标准品溶液,卡那霉素药物浓度的增加,试纸条T线的荧光强度逐渐减弱,呈现抑制状态,以无标准品抑制的荧光峰高值为F0值,相应浓度标准品抑制时的荧光峰高值为F值,以F/F0为纵坐标,以标准品浓度为横坐标,绘制标准曲线。利用Originpro 8.0软件拟合,曲线符合四参数方程,R2为0.9998,IC50值为24.8μg·L-1,线性范围(以20%—80%抑制率对应的浓度计算)为9.5—100μg·L-1,最低检出限(以10%抑制率对应的浓度计算)为5μg·L-1。准确度和精密度试验表明,以25、50和100μg·L-13个浓度添加水平,平均添加回收率为78.4%—92.7%,批内变异系数为10.8%—12.4%,符合残留检测的需求。特异性试验表明,试验建立的卡那霉素荧光微球免疫层析方法对其他常见的氨基糖苷类药物的交叉反应率均<1%,说明方法特异性良好。[结论]所建立方法快速灵敏、简单有效,特异性好,具有推广价值。

[Objective] Kanamycin is a common drug that is used for the treatment of bacterial diseases in dairy cattle,and its residue in milk can not be ignored.Fluorescent microsphere,as a new type of fluorescence tracer,has presented a unique advantage towards the detection of small molecules in recent years.This study introduces a fluorescent microsphere immunochromatographic quantitative method for detecting kanamycin in milk,which provides a rapid and effective screening means for monitoring kanamycin residues.[Method] Two coating antigens of KANA（KANA-GA-OVA1,KANA-I2-OVA2） were developed by glutaraldehyde method and Na IO4 method.After purification of anti-KANA monoclonal antibody by protein G immune affinity column,the antibody was coupled with fluorescent microspheres using EDC for the preparation of antibody-fluorescent microsphere conjugates（FM-mAbs）.The immunochromatographic strip test was conducted as follows：NC membrane was coated with kanamycin antigen as a reaction carrier,（FM-mAbs） incubated with the sample,and allowed to migrate from below.The drug in-sample and coating antigen compete with each other for the limited FM-mAbs,and the amount of drug was evaluated by detection of the fluorescence intensity of FM-mAbs combined with coating antigen.[Result] It shows that the fluorescence intensity of test line gradually weakened with increasing concentrations of KANA on the sensitivity test,when drug standard solutions were added into samples at 0,6.25,12.5,25,50,100 and 200 μg·L^-1.The F/F0 ratio was selected to express the competitive inhibition,where F0 and F are the fluorescence intensities respectively obtained from binding at zero and certain concentrations of the KANA standard.Standard curves were obtained by plotting F/F0 against the analyte concentration and fitted to a four-parameter logistic equation using Originpro 8.0 software.The dynamic range IC20-IC80 was calculated as 9.5-100 μg·L^-1,and the IC50 and LOD（IC10） were 24.8 and 5.0 μg·L^-1,respectively.When the blank samples were spiked at 25,50 and 100 μg·L^-1,the mean recoveries ranged from 78.4%-92.7%,with intra-assay coefficient of variations ranging from 10.8%-12.4%.The specificity of the method was evaluated by determining the cross-reactivity using other common aminoglycoside drugs.Negligible cross-reactivity（ 〈1%） was found for these other aminoglycosides.[Conclusion] The method is a rapid,sensitive,simple,effective,specific and suitable for promotion and application.