Preparation of Polyclonal Antibodies and Development of a Direct Competitive Enzyme-Linked Immunosorbent Assay To Detect Residues of Phenylethanolamine A in Urine Samples

Abstract

Phenylethanolamine A (PEAA) is a phenethanolamine member of the family of β-adrenergic agonists (β-agonists) compounds. To determine PEAA residues, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody produced with the immunogen PEAA-HSA conjugate. The antibody showed high sensitivity, where IC(50) and the limit of detection were 0.3 and 0.02 μg/L, respectively. The specificity of the assay was evaluated by the measurement of cross-reactivity of the antibody with 15 β-agonists compounds. The data demonstrated that the antibody was highly specific for PEAA, with negligible cross-reactivity (CR) with other β-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 μg/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.