Development of a monoclonal antibody-based direct competitive enzyme-linked immunosorbent assay for a new β-adrenergic agonist phenylethanolamine A

Abstract

A direct competitive enzyme-linked immunosorbent assay (dcELISA) based on a monoclonal antibody was developed for detecting a new β-adrenergic agonist phenylethanolamine A (PEAA). A PEAA derivative, PEAA–NH₂, was synthesized by hydrogenation using RANEY® nickel as a catalyst, before it was coupled to carrier proteins to generate PEAA–BSA, PEAA–OVA and PEAA–HRP via diazotization and sodium periodate methods, repectively. Three Balb/C mice were immunized with PEAA–BSA as the immunogen, and three clones stably producing antibodies against PEAA were acquired. Subsequently, a dcELISA method was established using the monoclonal antibody (clone 2H8) and PEAA–HRP. With the PEAA concentration range of 0.025–2.025 ng mL⁻¹, the IC₅₀ value was 0.204 ng mL⁻¹. The monoclonal antibody was highly specific for PEAA and its derivative (PEAA–NH₂), with minor cross-reactivity (CR) with ractopamine (CR = 8.3%) and negligible cross-reactivity with 15 other β-agonists compounds (CR < 0.1%). For sample testing, the LOD (limit of detection) values of blank samples (pork, swine liver, swine urine and feed) were shown to be 0.431, 0.455, 0.225 and 8.693 ng mL⁻¹, respectively; the corresponding LOQ (limit of quantification) values were 0.578, 0.647, 0.360 and 14.477 ng mL⁻¹, respectively. The recovery rates ranged from 82% to 120%. The coefficients of variation were below 13.33% and 10.53% for inter-assay and intra-assay, respectively. Furthermore, the dcELISA was validated by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method, and the result showed a high correlation coefficient between the two methods. Overall, the results suggested this dcELISA method with high sensitivity and specificity could be used to detect PEAA residues in real samples reliably.