Production of monoclonal antibody and development of enzyme-linked immunosorbent assay for kanamycin in biological matrices

Abstract

Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin–bovine γ-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC₅₀) for the MAbs were 2 and 5 ng ml^–1. One MAb (IC₅₀ = 2 ng ml^–1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml^–1 and the standard deviations were 0.2–4.4% for intra-assay and 0.6–4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma.