Development of an indirect competitive enzyme-linked immunosorbent assay for screening ethopabate residue in chicken muscle and liver

Abstract

Ethopabate (ETP) is a coccidiostat that is frequently used to prevent and treat coccidiosis in chickens. Illegal use and abuse of ETP can lead to drug residues in edible animal tissues, which present a potential health risk to consumers. To rapidly monitor ETP residues, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed that has simple sample preparation and clean-up. After immunogen preparation, inoculation and cell fusion, a monoclonal antibody (4G7) was obtained with the lgG2 isotype. The 4G7 antibody had the ability to specifically recognize ETP with an IC₅₀ value of 0.66 μg L⁻¹, it was found to have weak and insignificant cross-reactivity for some structure-related analogs. With the optimized ic-ELISA protocol, the detection limits of ETP were calculated as 0.21 μg kg⁻¹ and 0.34 μg kg⁻¹ in chicken muscle and liver samples, respectively. The recoveries ranged from 85.4% to 98.4% with a coefficient of variation of less than 15%. Furthermore, good correlations between the results of ic-ELISA and high-performance liquid chromatography demonstrated the reliability of the developed ic-ELISA. This proposed method is a rapid, sensitive and useful tool that offers a cost-effective alternative to current published procedures without any concession in the ability to detect ETP residues in edible animal tissues.