Salbutamol (SAL), a kind of β-adrenergic receptor agonists (β-agonists) which can enhance the lean meat-to-fat ratio, has been inhibited as an additive used in animal feeds for livestock production in many countries due to its harmful effect to the consumers. Herein we presented a sensitive and group-specific ELISA based on monoclonal antibody (mAb) for the determination of SAL in swine meat and liver samples. The SAL derivative containing carboxylic group was prepared by directly reacting SAL with succinic anhydride, then the derivative was linked to carrier proteins. Mice immunized with SAL-bovine serum albumin (BSA) conjugate were utilized for mAb generation. The hybridoma clone (1C3) specifically secreting mAb against SAL with the higher sensitivity was successfully obtained. Under optimal conditions, the IC50 and LOD values of the mAb-based ELISA for SAL were found to be 0.47 ng mL-1 and 0.049 ng mL-1, respectively, indicating high sensitivity of the assay. The mAb displayed high cross-reactivity (CR) with four β-agonists, e.g. clenbuterol (55%), terbutaline (44%), brombuterol (33%) and cimbuterol (134%) because these compounds contain the same tert-butyl group on the nitrogen atom like SAL has. The CR of the mAb with other β-agonists including cimaterol, clorprenaline, isoprenaline, neophryn, ractopamine and phenylethylamine A was less than 2.2% due to relatively large structure difference comparing to that of SAL. Swine meat and liver samples were spiked with different content of SAL and detected by ELISA. The recovery rates were respectively found in the range of 76.4-107.6% and 71.8-106.8%, with coefficients of variation less than 20%. Spiked samples were also analyzed by HPLC. A good correlation between two methods was obtained. These results demonstrated that the proposed ELISA was a feasible quantitative/screening method for SAL analysis in swine meat and liver samples. In addition, the recoveries results of the β-agonists which displayed the high CR values with mAb in an additional spiking experiment revealed the ELISA could be also as an alternative method for multiple β-agonists detection.

中文翻译：

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基于灵敏且基于组特异性单克隆抗体的间接竞争酶联免疫吸附测定法，用于测定猪肉和肝样品中的沙丁胺醇

沙丁胺醇（SAL）是一种β-肾上腺素能受体激动剂（β-激动剂），可提高瘦肉对脂肪的比例，由于其有害物质，在许多国家被禁止用作畜牧业动物饲料的添加剂对消费者的影响。本文中，我们介绍了一种基于单克隆抗体（mAb）的灵敏且特异性的ELISA方法，用于测定猪肉和肝样品中的SAL。通过使SAL与琥珀酸酐直接反应来制备含羧基的SAL衍生物，然后将该衍生物与载体蛋白连接。用SAL-牛血清白蛋白（BSA）结合物免疫的小鼠用于mAb产生。成功获得了以更高的敏感性特异性分泌针对SAL的mAb的杂交瘤克隆（1C3）。在最佳条件下 基于mAb的ELISA对SAL的IC50和LOD值分别为0.47 ng mL-1和0.049 ng mL-1，表明该方法具有很高的灵敏度。mAb与四种β激动剂（例如盐酸克伦特罗（55％），特布他林（44％），溴丁酚（33％）和香柏特罗（134％））表现出高交叉反应性（CR），因为这些化合物包含相同的叔丁基像SAL一样具有氮原子。与SAL相比，mAb与其他β-激动剂（包括cimaterol，clorprenaline，isoprenaline，neophrenn，ractopamine和苯乙胺A）的CR小于2.2％。猪的肉和肝样品中掺入不同含量的SAL，并通过ELISA检测。回收率分别在76.4-107.6％和71.8-106.8％范围内，变异系数小于20％。尖峰样品也通过HPLC分析。两种方法之间获得了良好的相关性。这些结果表明，所提出的ELISA是一种用于猪和肝脏样品中SAL分析的可行的定量/筛选方法。此外，在另一次加标实验中，显示具有mAb的高CR值的β激动剂的回收率结果表明ELISA也可以作为多种β激动剂检测的替代方法。