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

doi:10.3864/j.issn.0578-1752.2015.21.015

摘要/Abstract

摘要： 【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、200 µg·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24.8 µg·L^-1，线性范围（以20%—80%抑制率对应的浓度计算）为9.5—100 µg·L^-1，最低检出限（以10%抑制率对应的浓度计算）为5 µg·L^-1。准确度和精密度试验表明，以25、50和100 µg·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。

关键词: 荧光微球, 免疫层析, 卡那霉素, 残留, 牛奶

Abstract: 【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 NaIO₄ 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-IC₈₀was calculated as 9.5-100 µg?L^-1, and the IC₅₀and 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.

Key words: fluorescent microspheres, immunochromatography, kanamycin, residue, milk

徐飞，周洁，吴超，王建芬，丁双阳，李秀波. 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[J]. 中国农业科学, 2015, 48(21): 4358-4365.

XU Fei, ZHOU Jie, WU Chao, WANG Jian-fen, DING Shuang-yang, LI Xiu-bo. Development of a Microsphere-Based Fluorescence Immunochromatographic Assay for Detection of Kanamycin[J]. Scientia Agricultura Sinica, 2015, 48(21): 4358-4365.

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https://www.chinaagrisci.com/CN/Y2015/V48/I21/4358

参考文献

[1] 陈杖榴. 兽医药理学. 第二版. 北京：中国农业出版社, 2001.

Chen Z L. Veterinary Pharmacology. 2nd ed. Beijing: Chinese Agriculture Press, 2001. (in Chinese)

[2] 刘晓霞. 动物源性食品中氨基糖苷类抗生素残留分析方法研究 [D]. 长沙：湖南师范大学, 2011.

Liu X X. Study on the determination of aminoglycosides in foods of animal origin[D]. Changsha: Hunan Normal University, 2011. (in Chinese)

[3] 宓捷波, 郭振泉, 冯仁青. 现代抗体技术及其应用. 第1版. 北京：北京大学出版社, 2006.

Mi J B, Guo Z Q, Feng R Q. Modern Antibody Technique and Application. 1st ed. Beijing：Beijing University Press, 2006. (in Chinese)

[4] 康熙雄. 免疫胶体金技术临床应用. 第1版. 北京：军事医学科学出版社, 2010.

Kang X X. Immune Colloidal Gold Technique for Clinical Application. )1st ed. Beijing: Military Medical Science Press, 2010. (in Chinese

[5] 张贺秋. 生物医学实验技术系列丛书:胶体金标记探针与酶联免疫斑点技术. 第1版. 北京：军事医学科学出版社, 2012.

Zhang H Q. Biomedical Experiment Technology Series: Colloidal Gold Probe and Enzyme-Linked Immunospot Assay. 1st ed. Beijing: Military Medical Science Press, 2012. (in Chinese)

[6] Wong R C, Tse H Y. Lateral Flow Immunoassay. 1st ed. New York: Humana Press Inc. 2009.

[7] 胡杰, 刘白玲, 汪地强. 高通量药物筛选中的荧光微球. 现代化工, 2003(6):59-62.

Hu J, Liu B L, Wang D Q. Fluorescent particles in high - throughput screening assay.Modern Chemical Industry, 2003(6):59-62. (in Chinese)

[8] 于淼, 邹明强, 何昭阳. 高分子荧光微球在生物医学领域中的某些应用. 分析测试学报, 2006(3):115-119.

Yu M, Zou M Q, He S Y. Some application of high polymer fluorescent microsphere in biomedicine. Journal of Instrumental Analysis, 2006(3):115-119. (in Chinese)

[9] 韩文敏. 免疫检测荧光微球的制备和性能研究[D]. 武汉:华中科技大学，2008.

Han W M. Preparation and properties of immunoassay fluorescent microspheres[D]. Wuhan: Huazhong University of Science and Technology, 2008. (in Chinese)

[10] Thiollet S, Higson S, White N, Morgan S L. Investigation and development of quantum dot-encoded microsphere bioconjugates for DNA detection by Flow Cytometry. Journal of Fluorescence, 2012, 22(2):685-697.

[11] Czeh A, Mandy F, Feher-Toth S, Torok L, Milk Z, Koszegi B, Lustyik G. A flow cytometry based competitive fluorescent microsphere immunoassay (CFIA) system for detecting up to six mycotoxins. Journal of Immunological Methods, 2012, 384(1/2):71-80.

[12] Guo Y R, Tian J, Liang C Z, Zhu G N, Gui W J. Multiplex bead-array competitive immunoassay for simultaneous detection of three pesticides in vegetables. Microchimica Acta, 2013, 180(5/6):387-395.

[13] Fuller K M, Arriaga E A. Analysis of individual acidic organelles by capillary electrophoresis with laser-induced fluorescence detection facilitated by the endocytosis of fluorescently labeled microspheres. Analytical Chemistry, 2003, 75 (9):2123-2130.

[14] 宋秀玲. 多酸基荧光检测微球的制备及布鲁氏菌检测方法的研究[D]. 长春：吉林大学, 2013.

Song X L. Preparation of POMs-based fluorescent detection microspheres and study on the detection method of Brucella [D]. Changchun: Jilin University, 2013. (in Chinese)

[15] 董香梅. 单核细胞增生李斯特菌单克隆抗体制备及荧光微球免疫层析试纸条的研制[D]. 南昌:南昌大学, 2013.

Dong X M. Preparation of monoclonal antibodies and fluorescent beads based immnochroniatographic strip for detection of Listeria monocytogenes [D]. Nanchang：Nanchang University, 2013. (in Chinese)

[16] Xie Q Y, Wu Y H, Xiong Q R, Xu H Y, Xiong Y H, Liu K, Jin Y, Lai W H. Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays. Biosensors and Bioelectronics, 2014, 54:262-265.

[17] 李姮. 伏马菌素B1和脱氧雪腐镰刀菌烯醇的免疫层析检测方法研究[D]. 北京：中国农业大学, 2013.

Li H. Study on immunochromatographic assay of fumonisin B1 and deoxynivalenol [D]. Beijing: China Agricultural University, 2013. (in Chinese)

[18] Bian S M, Chu X G, Jin Y, Xing S G, Zhang Y, Hu H B. A novel microsphere-based fluorescence immunochromatographic assay for monitoring cefalexin residues in plasma, milk, muscle and liver. Analytical Methods, 2013, 5(22): 6441-6448.

[19] 崔浩. 莱克多巴胺荧光微球免疫层析快速检测方法的建立[D]. 广州: 暨南大学, 2012.

Cui H. Development of an immunochromatography assay with fluorescent microspheres for the rapid detection of ractopamine [D]. Guangzhou: Jinan University, 2012. (in Chinese)

[20] Chen R, Li H, Zhang H, Zhang S X, Shi W M, Shen J Z, Wang Z H. Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk. Analytical and Bioanalytical Chemistry, 2013, 405(21):6783-6789.

[21] Zhou J, Zhu K, Xu F, Wang W J, Jiang H Y, Wang Z H, Ding S Y. Development of a microsphere-based fluorescence immunochromatographic assay for monitoring lincomycin in milk, honey, beef, and swine urine. Journal of Agricultural Food Chemistry, 2014, 62(49):12061-12066.

[22] Chen Y Q, Wang Z Q, Wang Z H, Tang S S, Zhu Y, Xiao X L. Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramacin in swine tissues. Journal of Agricultural and Food Chemistry, 2008, 56(9):2944-2952.

[23] Probes M. Working With FluoSpheres® Fluorescent Microspheres. [2014-4-8]. http://tools.lifetechnologies.com/content/sfs/manuals/mp05001. pdf.

[24] 顾春峰, 兰秀风, 于银山, 卢礼萍. 牛奶水溶液的荧光光谱研究. 光子学报, 2012, 41(1):107-111.

Gu C F, Lan X F, Yu Y S, Lu L P. Fluorescence spectrum of milk solution. Acta Photononica Sinica, 2012, 41(1):107-111. (in Chinese)