Novel ractopamine–protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed

Abstract

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.

概 要

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–BSA) 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。