Preparation of Polyclonal Antibodies and Development of a Direct Competitive Enzyme-Linked Immunosorbent Assay To Detect Residues of Phenylethanolamine A in Urine Samples
Preparation of Polyclonal Antibodies and Development of a Direct Competitive Enzyme-Linked Immunosorbent Assay To Detect Residues of Phenylethanolamine A in Urine Samples
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DOI：
10.1021/jf3036066
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发表时间：
2012-11-21
影响因子：
6.1
通讯作者：
Xu, Shixin
中科院分区：
文献类型：
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作者：
Bai, Yuhui;Liu, Zhihong;Xu, Shixin
苯乙醇胺A（PEAA）是β-肾上腺素能激动剂（β-激动剂）化合物家族的苯乙醇胺成员。为了确定PEAA残留，我们建立了一个快速的直接竞争酶联免疫吸附试验（EIASA）使用的多克隆抗体与免疫原PEAA-HSA缀合物。该抗体具有较高的灵敏度，IC_（50）为0.3 μ g/L，检出限为0.02 μ g/L。通过测量抗体与15种β-激动剂化合物的交叉反应性，评价了测定的特异性。数据表明，该抗体对PEAA具有高度特异性，与包括莱克多巴胺（CR为0.3%）在内的其他β-激动剂化合物的交叉反应性（CR）可忽略不计（CR <0.1%）。回收率范围为81%至110%，表明当应用于真实的样品时，该测定法具有相对良好的平行性和准确度。空白尿样的检出限为0.5 μ g/L。试验内和试验间的变异系数分别低于18%和20%，证明精密度水平可接受。ELISA法和UPLC-MS/MS法对尿样的检测结果基本一致。从实用的角度来看，原型试剂盒可有利地用于筛查大组尿样，并且所采用的试剂盒即使在用于控制药物非法使用的常规应用中也具有可靠性。
Phenylethanolamine A (PEAA) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists) compounds. To determine PEAA residues, we established a rapid direct competitive enzyme linked immunosorbent assay (EIASA) using a polyclonal antibody produced with the immunogen PEAA-HSA conjugate. The antibody showed high sensitivity, where IC50 and the limit of detection were 0.3 and 0.02 mu g/L, respectively. The specificity of the assay was evaluated by the measurement of cross-reactivity of the antibody with 15 beta-agonists compounds. The data demonstrated that the antibody was highly specific for PEAA, with negligible cross-reactivity (CR) with other beta-agonists compounds (CR < 0.1%) including ractopamine (CR is 0.3%). Recovery rates ranged from 81% to 110%, indicating relatively good parallelism and accuracy of the assay when applied to real samples. The detection limit in blank urine samples was 0.5 mu g/L. The coefficient of variation was below 18% and 20% for intra-assay and inter-assay, respectively, demonstrating an acceptable level of precision. Largely consistent results were obtained for the urine samples by ELISA and UPLC-MS/MS methods. From a practical point of view, the prototype kit could be advantageously used for the screening of large groups of urine samples, and the kit employed has reliability even in routine application for the control of the illegal use of the drug.