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目 的:开发一种新型简单快速的克莱多巴胺-蛋白质载体结合方法,用于侧向流试纸条检测动物饲料.创新点:基于曼尼希反应的克莱多巴胺-牛血清蛋白(RAC-B SA)结合方法,具有一步性、简单、快速,且所需试剂少的优点.方 法:通过曼尼希反应产生RAC-BSA结合物,制备竞争型侧向流试纸条.将RAC标记于猪饲料中,以体积比10∶90的甲醇:电泳缓冲液作为提取缓冲液,在25 min内完成样品制备.通过裸眼观察定性评估RAC-BSA结合物的免疫应答以及检测灵敏性,通过智能手机和电脑软件(ImageJ)进行半定量分析.结 论:在最佳条件下,侧向流试纸条检测的检出限(LOD)在5 min裸眼评估下为1 ng/g;半定量分析中,其线性范围为0.075～0.750 ng/g,计算的LOD为0.10 ng/g,定量限为0.33 ng/g,且相关性较好(R2=0.992).回收率为96.4％～103.7％,同实验内及不同实验间的相对标准偏差为2.5％～3.6％.通过与酶联免疫吸附法对比,该侧向流试纸条检测结果具有较高的准确性和特异性.因此,通过曼尼希反应制备RAC-BSA结合物是一步的简单的结合法,可用于侧向流试纸条检测法.“,”In this work,a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction,with a mole coupling ratio for RAC-BSA of 9:1.The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays.RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed.For sample preparation,RAC was spiked in swine feed purchased from the local markets in Thailand,and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer.The procedures for sample preparation were completed within 25 min.Under optimal conditions,the limit of detection (LOD),assessed by the naked eye within 5 min,was found to be 1 ng/g.A semi-quantitative analysis was also conducted using a smart phone and computer software,with a linearity of 0.075-0.750 ng/g,calculated LOD of 0.10 ng/g,calculated limit of quantitation of 0.33 ng/g,and good correlation of 0.992.The recoveries were found in the range of 96.4％-103.7％ with a relative standard deviation of 2.5％-3.6％ for intra-and inter-assays.Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy.Furthermore,this strip test exhibited highly specific RAC detection without cross reactivity with related compounds.Therefore,the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.