Neamin as an Immunogen for the Development of a Generic ELISA Detecting Gentamicin, Kanamycin, and Neomycin in Milk

Abstract

A broad-specific ELISA using one antibody preparation for the detection of gentamicin, kanamycin, and neomycin in milk is reported for the first time. For the immunization of rabbits, neamin was used as the generic hapten on the basis of the facts that it is a two-ring fragment of neomycin and, in shape and charge, it resembles parts of kanamycin and gentamicin. Neamin was linked to the preactivated carrier protein keyhole limpet hemocyanin by EDC coupling. The specificity and sensitivity of the polyclonal antibodies for the aminoglycoside antibiotics were tested in a competitive assay using homologous and heterologous conjugates coupled by various conjugation procedures as the ELISA solid phase. In contrast to the homologous assay recognizing only neomycin, the heterologous assay could be used for the detection of the whole subclass of deoxystreptamin antibiotics in buffer and raw milk. Gentamicin, kanamycin, and neomcyin were detected in artificially contaminated undiluted raw milk (without sample pretreatment) with 50% inhibition levels at 9, 21, and 113 ng mL(-)(1), respectively. Neomycin levels were also measured in milk samples obtained from a cow suffering from mastitis and treated with an antibiotic cocktail including neomycin. Levels below the EU maximum residue levels for deoxystreptamin antibiotics could readily be measured in this generic ELISA.

Analysis of kanamycin A in human plasma and in oral dosage form by derivatization with 1-naphthyl isothiocyanate and high-performance liquid chromatography

“…Immunoassays [5,6], spectrophotometric analysis [7,8], HPLC techniques [9 -15], and CE [16 -18] have been used for the determination of kanamycin A in various sample matrices. In immunoassays, cross-reactions are sometimes noted and the reagents are expensive and would also be inappropriate for determination of the potential presence of degradation products or impurities.…”

Broad-spectrum protein biosensors for class-specific detection of antibiotics

“…Several technologies are currently available for detection of specific antibiotics, including (i) chromatographic methods often used in combination with mass spectrometry (Schenck and Callery, 1998), (ii) antibiogram tests revealing antimicrobial activities on specific indicator strains (Korsrud et al, 1998), and (iii) immunodetection based on antibodies specific for particular antibiotic structures (Lee et al, 2001;Loomans et al, 2003). Unfortunately, (i) most of these antibiotic detection strategies are hardwareintensive, (ii) provide limited readout information, or (iii) turn out to be very specific for only a particular antibiotic structure.…”

Engineering an Allosteric Binding Site for Aminoglycosides into TEM1‐β‐Lactamase

“…The submicromolar affinities of the first three aminoglycosides are not very different from the affinities of monoclonal antibodies for kanamycin (20-40 nm), as calculated from the data of Watanabe [22] or Loomans. [23] Affinities for oligosaccharides or similar compounds are rarely in the nanomolar range, because the hydrogen bonds contributing to binding are solvated by water in the free protein and ligand. [28,29] As far as the aminosulfonic acids are concerned, BES is a better inhibitor than MES, which is in turn superior to taurine.…”

“…[21] Aminoglycosides have been used extensively by veterinary practitioners, and their presence in foodstuffs is a growing source of concern, and hence must be monitored. [22,23] Furthermore, as they are rather hydrophilic compounds, it is unlikely they would bind to the enzyme primarily by nonspecific hydrophobic interactions. We show that this enzyme is activated by the binding of aminoglycosides.…”