J Vet Sci.  2006 Jun;7(2):111-117. 10.4142/jvs.2006.7.2.111.

Development of immunoassays for the detection of kanamycin in veterinary fields

• Jin Y
• Jang JW
• Han CH
• Lee MH

• KMID: 1059194
• DOI: http://doi.org/10.4142/jvs.2006.7.2.111

Abstract

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.

Keyword

competitive direct ELISA; immunochromatographic assay; kanamycin; monoclonal antibody

MeSH Terms

Animals
Anti-Bacterial Agents/*analysis
Antibodies, Monoclonal
Chromatography/methods/veterinary
Enzyme-Linked Immunosorbent Assay/methods/*veterinary
Kanamycin/*analysis
Mice
Milk/*chemistry
Rabbits

Figure

• 

  Fig. 1 Determination of monoclonal antibody isotype. Rabbit antisera specific for mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, and lambda light chain were added to each well, and detected with goat anti-rabbit IgG conjugated with peroxidase. The negative control included only pre-immune serum.

• 

  Fig. 2 Standard curve of kanamycin in PBS (○), rabbit plasma (●), and bovine milk (▲) constructed through competitive direct ELISA. B and B0 are the absorbance at 490 nm in the presence or absence of free kanamycin. Each value shows the mean (±SD) of B/B0 (n = 4).

• 

  Fig. 3 Depletion profile of kanamycin in rabbit plasma after intramuscular administration of kanamycin. Kanamycin was administered intramuscularly to rabbits at 20 mg/kg/day for 3 consecutive days. Blood samples were collected from the ear vein of each rabbit 2, 4, 6, 8, 10, and 12 h after the last injection of kanamycin. Plasma samples were diluted 10-fold in PBS and subjected to the competitive direct ELISA. Each value shows the mean (±SD) of kanamycin concentration (n = 4).

• 

  Fig. 4 Cross-reactivity of the monoclonal antibody of kanamycin with aminoglycosides in immunochromatographic assay. Three microgram of kanamycin-BSA, gentamicin-BSA, neomycin-BSA, or streptomycin-BSA conjugate was applied to each strip of a nitrocellulose membrane. After the gold-labeled antibody moved up the membrane, the intensity of the red color band on each membrane strip was observed.

• 

  Fig. 5 Immunochromatographic assay for the detection of kanamycin. A series of dilutions (0, 0.5, 1, 2, 4, 6, and 8 ng/ml) of kanamycin were prepared in PBS (A), rabbit plasma (B), and bovine milk (C).

• 

  Fig. 6 Comparison of immunochromatographic assay with competitive direct ELISA. Kanamycin was administered intramuscularly to rabbits at 20 mg/kg/day for 3 consecutive days as described in Materials and Methods. Blood samples were collected from rabbits 2, 4, 6, 8, 10, and 12 h after the last injection of kanamycin. Plasma samples were subjected to immunochromatographic assay. Subsequent competitive direct ELISA determined the kanamycin concentration in plasma samples.

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