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Issue 15, 2014
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Development of a monoclonal antibody-based direct competitive enzyme-linked immunosorbent assay for a new β-adrenergic agonist phenylethanolamine A

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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.

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

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Article information


Submitted
18 Mar 2014
Accepted
05 May 2014
First published
12 May 2014

Anal. Methods, 2014,6, 5942-5950
Article type
Paper

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

L. Zhang, Y. Gong, M. Zhang, X. Xi, M. Li, Z. Chen, X. Yu and Y. Zhou, Anal. Methods, 2014, 6, 5942
DOI: 10.1039/C4AY00682H

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