Development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk

ABSTRACT The flunixin residues in foods of animal origin could pose hazards for human health. In this paper, a broad-selectivity polyclonal antibody was obtained using 5-hydroxyflunixin coupled with bovine serum albumin as the immunogen. The semi-inhibitory concentration (IC50) of the polyclonal antibody was 1.43？ng/mL for flunixin and 0.29？ng/mL for 5-hydroxyflunixin, respectively, which were far below the maximum residue limits set by the United States, the European Union and China. The limits of detection were calculated to be 2.98？μg/kg for flunixin in bovine muscle and 0.78？μg/L for 5-hydroxyflunixin in milk. In spike and recovery tests, the average recovery ranged from 83% to 105%, with a satisfying coefficient of variance ranging from 5.8% to 11.3%. Furthermore, the ELISA method was validated by a well-established LC-MS/MS method. These results proved that the established method is suitable for flunixin and 5-hydroxyflunixin residue analysis