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.

References

1. Analytical Methods for Residual Veterinary Drugs in Food, ed. T. Hayama and Y. Kido, Kindai, Tokyo, 1994, pp. 65–76(in Japanese).
2. D. M. Barends, J. S. F. van der Sandt and A. Hulshoff, J. Chromatogr., 1980, 182, 201.
3. D. M. Barends, C. L. Zwaan and A. Hulshoff, J. Chromarogr., 1981, 222, 316.
4. D. M. Barends, C. L. Zwaan and A. Hulshoff, J. Chromatogr., 1981, 225, 417.
5. M. Preu, D. Guyot and M. Petz, J. Chromatogr. A, 1998, 818, 95.
6. L. G. McLaughlin, J. D. Henion and P. J. Kijak, Biol. Mass Spectrom., 1994, 23, 417.
7. T. Kitagawa, K. Fujiwara, S. Tomonoh, K. Takahashi and M. Koida, J. Biochem., 1983, 94, 1165.
8. J. D. Place, S. G. Thompson, J. F. Burd, C. Molinaro, R. A. Ott and F. C. Jensen, Hybridoma, 1984, 3, 187.
9. P. Schnappinger, E. Usleber, E. Maltbauer and G. Terplan, Food Agric. Immunol., 1993, 5, 67.
10. J. Ara, Z. Gans, R. Sweeney and B. Wolf, J. Clin. Lab. Anal., 1995, 9, 320.
11. B. F. Erlanger, Methods Enzymol., 1980, 70, 85.
12. M. Kohler and C. Milstein, Nature (London), 1975, 256, 495.
13. H. Watanabe, A. Satake, M. Matsumoto, Y. Kido, A. Tsuji, K. Ito and M. Maeda, Analyst, 1998, 123, 2573.
14. O. J. Boison and D. J. MacNeil, in Chemical Analysis for Antibiotics Used in Agriculture, ed. H. Oka, H. Nakazawa, K. Harada and D. J. MacNeil, AOAC International, Arlington, VA, 1995, pp. 77–119.
15. M. Matsumoto, K. Tsunematsu, A. Tsuji and Y. Kido, Anal. Chim. Acta, 1997, 346, 207.