Preparation of polyclonal antibody and development of a biotin-streptavidin-based ELISA method for detecting kanamycin in milk and honey

Abstract

Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects. Here we reported the preparation of polyclonal antibody and the development of an indirect competitive biotin-streptavidin-amplified-based enzyme-linked immunosorbent assay(BA-ELISA) that can sensitively and specifically detect kanamycin residues in milk and honey. The immunogen and coating antigen were synthesized by covalently linking kanamycin to carrier proteins using the carbodiimide method. The anti-kanamycin polyclonal antibodies were obtained from immunized rabbits. The key assay parameters were investigated and optimized. The results show that under optimum conditions, the limit of detection for kanamycin is 0.07 ng/mL and the IC₅₀ is 6.48 ng/mL. Cross-reactivity values of the antibody with four kanamycin analogues are all <1%. Trace amounts of kanamycin in milk and honey samples can be detected by this novel BA-ELISA method successfully with satisfactory recoveries of 91.0%—103.3%. The developed protocol was also validated against liquid chromatography-mass spectrometry, returning a significant correlation. These results indicate that BA-ELISA is a viable option for monitoring kanamycin residues in milk and honey.

Keywords

Kanamycin / Biotin-streptavidin-amplified system / Enzyme-linked immunosorbent assay(ELISA) / Milk / Honey

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