Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

  • Jin, Yong (Department of Biochemistry, College of Veterinary Medicine, Seoul National University) ;
  • Jang, Jin-Wook (Department of Biochemistry, College of Veterinary Medicine, Seoul National University) ;
  • Lee, Mun-Han (Department of Biochemistry, College of Veterinary Medicine, Seoul National University) ;
  • Han, Chang-Hoon (Department of Biochemistry, College of Veterinary Medicine, Seoul National University)
  • Received : 2005.03.17
  • Accepted : 2005.06.28
  • Published : 2005.10.01


Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities ($CR_{50}$) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/$B_0$ ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/$B_0$ ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life ($t_{1/2}$) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals.


Monoclonal Antibody;Gentamicin;Aminoglycosides;Competitive Direct ELISA


Supported by : Seoul National University


  1. Berkowitz, D. B. and D. W. Webert. 1986. Enzyme immunoassaybased survey of prevalence of gentamicin in plasma of marketed swine. J. AOAC Int. 69:437-441.
  2. Fourmy, D., S. Yoshizawa and J. D. Puglisi. 1998. Paromomycin binding induces a local conformational change in the A-site of 16S rRNA. J. Mol. Biol. 277:333-345.
  3. Kitagawa, T., K. Fujiwara, S. Tomonoh, K. Takahashi and M. Koida. 1983. Enzyme immunoassays of kanamycin group antibiotics with high sensitivities using anti-kanamycin as a common antiplasma: reasoning and selection of a heterologous enzyme label. J. Biochem. 94:1165-1172.
  4. Ploczekove, C. and O. Foldes. 1992. Immunochemical determination of gentamicin in serum. III. The competitive ELISA. Cesk Epidemiol Mikrobiol Imunol. 41(6):346-354.
  5. Lantz, C. H., D. J. Lawrie, F. G. Witebsky and J. D. Maclowry. 1980. Evaluation of plasma gentamicin assay procedure for a clinical microbiology laboratory. J. Clin. Microbiol. 10:583-589.
  6. Stead, D. A. and R. M. E. Richards. 1997. Sensitive highperformance liquid chromatographic assay for aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake. J. Chromatogr. B 693:415-421.
  7. Isoherranen, N. and S. Soback. 1999. Chromatographic methods of analysis of aminoglycoside antibiotics. J. AOAC Int. 82:1017-1045.
  8. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.
  9. Hewitt, W. L. 1974. Gentamicin: toxicity in perspective. Postgrad. Med. J. 7:55-61.
  10. European Union regulation no. 1140/96, L151 (26-6-1996).
  11. Haasnoot, W., P. Stouten, G. Cazemier, A. Lommen, J. F. M. Nouws and H. J. Keukens. 1999. Immunochemical detection of aminoglycosides in milk and kidney. Analyst 124:301-305.
  12. Hanes, S. D. and V. L. Herring. 2001. Gentamicin enzyme-linked immunosorbent assay for microdialysis samples. Ther. Drug. Monit. 23(6):689-93.
  13. Tsay, Y. G., L. Wilson and E. Keefe. 1980. Quantitation of plasma gentamicin concentration by a solid-phase immunofluorescence method. Clin. Chem. 26:1610-1612.
  14. Stead, D. A. and R. M. E. Richards. 1996. Sensitive fluorimetric determination of gentamicin sulfate in biological matrices using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-phase highperformance liquid chromatography. J. Chromatogr. B 675:295-302.
  15. Place, J. D., S. G. Thompson, H. M. Clements, R. A. Ott and F. C. Jensen. 1983. Gentamicin substrate-labeled fluorescent immunoassay containing monoclonal antibody. Antimicrob. Agents Chemother. 8:246-251.
  16. Cleveland, C. B., D. E. Francke, W. M. Heller, J. A. Kepler, G. P. Provost and M. J. Reilly. 1990. Anti-Infective Agents. In AHFS Drug Information 90. American society of hospital pharmacists press, Bethesda, MD, pp. 51-67.
  17. Rosner, A. and H. Aviv. 1980. Gentamicin bioautography assay vs. the microbiological disk test. J. Antibiot. 6:600-603.
  18. Riviere, J. E. and J. W. Spoo. 2001. Aminoglycoside antibiotics. In Veterinary Pharmacology and Therapeutics, 8th edition; (Ed. H. R. Adams); Iowa State University Press, Iowa, pp. 841-867.
  19. Harlow, E. D. and D. Lane. 1988. Monoclonal antibodies. In Antibodies: A Laboratory Manual. Cold spring harbor laboratory, New York, NY, pp. 196-214.
  20. Posyniak, A., J. Zmudzki and J. Niedzielska. 2001. Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography. J. Chromatogr A. 914:59-66.
  21. Reynolds, J. E. F. 1993. Martindale: The extra pharmacopoeia 30th edition. The pharmaceutical press, London, UK, pp. 109-113.
  22. Bauminger, S. and M. Wilchek. 1980. The use of carbodiimides in the preparation of immunizing conjugates. Method Enzymol. 70:151-159.
  23. Abou-Zeid, A. A. and Y. M. Shehata. 1977. Gentamicins. Zbl. Bakt. Parasit. Infekt. Hyg. 132:97-108.
  24. Lewis, J. E., J. C. Nelson and H. A. Elder. 1972. Radioimmunoassay of an antibiotic: gentamicin. Nature New Biology 239:214-216.
  25. Graham, A. E., E. Speicher and B. Williamson. 1997. Analysis of gentamicin sulfate and a study of its degradation in dextrose solution. J. Pharmaceut. Biomed. 15:537-543.
  26. Ramsden, R. T., P. Wilson and W. P. R. Gibson. 1980. Immediate effects of intravenous tobramycin and gentamicin on human cochlear function. J. Laryngol. Otol. 94:521-531.
  27. Loomans, E. E. M. G., J. V. Wiltenburg, M. Koets and A. V. Amerongen. 2003. Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin, and neomycin in milk. J. Agric. Food Chem. 51:587-593.
  28. Ren, Y. G., J. Martinez, L. A. Kirsebom and A. Virtanen. 2002. Inhibition of klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides. RNA 8:1393-1400.
  29. Dixon, D. E., R. L. Warner, B. P. Ram, L. P. Hart and J. J. Pestka. 1987. Hybridoma cell line production of specific monoclonal antibody to the mycotoxins zearalenone and $\alpha$-zearalenol. J. Agric. Food Chem. 35:122-126.

Cited by

  1. ELISA-based detection of gentamicin and vancomycin in protein-containing samples vol.4, pp.1, 2015,