Simultaneous detection of tylosin and tilmicosin in honey using a novel immunoassay and immunochromatographic strip based on an innovative hapten

Abstract

ABSTRACTTylosin (TYL) and tilmicosin (TIM) are macrolide antibiotics, and maximum residue limits (MRLs) have been set to control their illegal usage in food. We developed a sensitive indirect competitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip (ICS) test to simultaneously detect TYL and TIM based on an innovative hapten (TYL-carboxymethoxylamine hemihydrochloride). The monoclonal antibody 2B3 was obtained with the isotype IgG1, and TYL and TIM had half maximal inhibitory concentrations of 0.57 and 2.10 ng/ml, respectively. The cross-reactivity values were 100%, 27.57%, 97.43%, and 62.42% for TYL, TIM, desmycosin, and acetylisovaleryl tylosin, respectively. By optimizing, the standard dilution buffer was based on 0.8% NaCl, pH 7.4 and 5% acetonitrile. The recoveries ranged from 89.37% to 112.00% in the honey samples, which suggests that the assay would be reliable in the analysis of real honey samples. In addition, an ICS was developed for qualitative, semi-quantitative, and spot detection analysis, with cut-off values of 5 and 15 ng/ml for TYL and TIM in phosphate-buffered saline, and 10 and 20 ng/ml for TYL and TIM in honey, respectively. The results show that these assays can be used to analyze real samples with the strictest MRL being 50 ng/ml for both TYL and TIM.