Abstract: 【Objective】 Kanamycin is a common drug that is used for the treatment of bacterial diseases in dairy cattle, and its residue in milk can not be ignored. Fluorescent microsphere, as a new type of fluorescence tracer, has presented a unique advantage towards the detection of small molecules in recent years. This study introduces a fluorescent microsphere immunochromatographic quantitative method for detecting kanamycin in milk, which provides a rapid and effective screening means for monitoring kanamycin residues. 【Method】 Two coating antigens of KANA (KANA-GA-OVA₁, KANA-I2-OVA₂) were developed by glutaraldehyde method and NaIO₄ method. After purification of anti-KANA monoclonal antibody by protein G immune affinity column, the antibody was coupled with fluorescent microspheres using EDC for the preparation of antibody-fluorescent microsphere conjugates (FM-mAbs). The immunochromatographic strip test was conducted as follows: NC membrane was coated with kanamycin antigen as a reaction carrier, (FM-mAbs) incubated with the sample, and allowed to migrate from below. The drug in-sample and coating antigen compete with each other for the limited FM-mAbs, and the amount of drug was evaluated by detection of the fluorescence intensity of FM-mAbs combined with coating antigen. 【Result】 It shows that the fluorescence intensity of test line gradually weakened with increasing concentrations of KANA on the sensitivity test, when drug standard solutions were added into samples at 0, 6.25, 12.5, 25, 50, 100 and 200 µg?L^-1. The F/F₀ ratio was selected to express the competitive inhibition, where F₀ and F are the fluorescence intensities respectively obtained from binding at zero and certain concentrations of the KANA standard. Standard curves were obtained by plotting F/F₀ against the analyte concentration and fitted to a four-parameter logistic equation using Originpro 8.0 software. The dynamic range IC₂₀-IC₈₀ was calculated as 9.5-100 µg?L^-1, and the IC₅₀ and LOD (IC₁₀) were 24.8 and 5.0 μg?L^-1, respectively. When the blank samples were spiked at 25, 50 and 100 µg?L^-1, the mean recoveries ranged from 78.4%-92.7%, with intra-assay coefficient of variations ranging from 10.8%-12.4%. The specificity of the method was evaluated by determining the cross-reactivity using other common aminoglycoside drugs. Negligible cross-reactivity (＜1%) was found for these other aminoglycosides. 【Conclusion】 The method is a rapid, sensitive, simple, effective, specific and suitable for promotion and application.

Key words: fluorescent microspheres, immunochromatography, kanamycin, residue, milk