Tylosin (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 (TYLcarboxymethoxylamine 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 cutoff values of 5 and 15 ng/ml for TYL and TIM in phosphatebuffered 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.

Kunitz trypsin inhibitor (KTI) is a predominant anti-nutritional factor in soybean. The objective of this study was to develop a sandwich enzyme-linked immunosorbent assay and immunochromato graphic strip (ICS) method for the semi-quantification of KTI in soybean. The limit of detection was 0.1481 ng/ml, and the linear dynamic range was 0.2-20 ng/ml. KTI recovery was 98.40-110.66% from KTI-spiked milk samples, with coefficients of variation of 4.41-10.62%. Based on ICS results, KTI had cutoff values of 5 ng/ ml and 2.5 ng/ml in 0.05% Tween-added phosphate-buffered saline and milk, respectively. ICS has potential applications for the fast detection of KTI.