Immunoassay Analysis of Kanamycin in Serum Using the Tobramycin Kit.

Kanamycin is one of the aminoglycosides used in the treatment of multidrug-resistant tuberculosis. Blood concentrations of kanamycin are predictive for the treatment efficacy and the occurrence of side effects, and dose adjustments can be needed to optimize therapy. However, an immunoassay method for the quantification of kanamycin is not commercially available. We modified the existing tobramycin immunoassay to analyze kanamycin. This modified method was tested in a concentration range of 0.3 to 80.0 mg/liter for inaccuracy and imprecision. In addition, the analytical results of the immunoassay method were compared to those obtained by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method using Passing and Bablok regression. Within-day imprecision varied from 2.3 to 13.3%, and between-day imprecision ranged from 0.0 to 11.3%. The inaccuracy ranged from -5.2 to 7.6%. No significant cross-reactivity with other antimicrobials and antiviral agents was observed. The results of the modified immunoassay method were comparable with the LC-MS/MS analytical outcome. This new immunoassay method enables laboratories to perform therapeutic drug monitoring of kanamycin without the need for complex and expensive LC-MS/MS equipment.

Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles.

Herein, we report the development of extremely sensitive sandwich assay of kanamycin using a combination of anti-kanamycin functionalized hybrid magnetic (Fe3O4) nanoparticles (MNPs) and 2-mercaptobenzothiazole labeled Au-core@Ag-shell nanoparticles as the recognition and surface-enhanced Raman scattering (SERS) substrate, respectively. The hybrid MNPs were first prepared via surface-mediated RAFT polymerization of N-acryloyl-L-glutamic acid in the presence of 2-(butylsulfanylcarbonylthiolsulfanyl) propionic acid-modified MNPs as a RAFT agent and then biofunctionalized with anti-kanamycin, which are both specific for kanamycin and can be collected via a simple magnet. After separating kanamycin from the sample matrix, they were sandwiched with the SERS substrate. According to our experimental results, the limit of detection (LOD) was determined to be 2pg mL(-1), this value being about 3-7 times more than sensitive than the LOD of previously reported results, which can be explained by the higher SERS activity of silver coated gold nanoparticles. The analysis time took less than 10min, including washing and optical detection steps. Furthermore, the sandwich assay was evaluated for investigating the kanamycin specificity on neomycin, gentamycin and streptomycin and detecting kanamycin in artificially contaminated milk.

Immunosensor based on magnetic relaxation switch and biotin-streptavidin system for the detection of Kanamycin in milk.

A rapid, sensitive, and simple immunosensor was developed for the detection of Kanamycin (KM) in milk. This immunosensor is based on magnetic relaxation switch (MRS) assay and biotin-streptavidin system (B-SA system). The target analyte (KM) competed with those on the surface of the superparamagnetic iron oxide (SPIO) nanoparticles and hence affected the formation of SPIO aggregates. The dispersed and aggregated states of SPIO can modulate the spin-spin relaxation time (T(2)) of the neighboring water molecule. T(2) was then changed as an effect of the target analyte. The B-SA system was used to amplify the SPIO binding, thus enhance the sensitivity. The detection working was 1.5 to 25.2ng mL(-1) and limit of detection (LOD) was determined to be 0.1ng mL(-1). The LOD of the immunosensor decreased tenfold, and its analysis time (45min) was much shorter than that of enzyme-linked immunosorbent assay (6h to 8h). The average recoveries of the KM at various spiking levels ranged from 80.2% to 85.6% with a relative standard deviation (RSD) below 4.0%. The results showed that the MRS immunosensor was a promising platform for the determination of small molecular residues because of its high sensitivity, specificity, homogeneity, and speed.

[Monoclonal antibody based enzyme-linked immunosorbent assay for aminoglycoside antibiotic kanamycin in foodstuff].

Monoclonal antibodies to aminoglycoside antibiotic kanamycin (KM) were raised as a result of mice complex immunization with glutaraldehyde conjugates BSA with KM, tobramycin (TM) and gentamicin. Using antibodies an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows to determine antibiotic up to 1.2 ng/ml in water solutions, milk and eggs and up to 2.5 ng/ml in honey. The recovery rate from these products spiked with KM was 83, 84 and 96% respectively. The assay of KM based on homologous and heterologous solid-phase conjugates were estimated. The cross-reactivity with TM could vary from 7 to 54%. The same indexes for of amikacin were more constant and reached 7-8%. The other aminoglycosides showed no inhibitory activity.

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

Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin-bovine gamma-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.

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay.

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

Contact dermatitis to antituberculosis drugs.

The literature has been reviewed for contact dermatitis occurring to antituberculosis agents. Of the 12 known drugs, 6 (isoniazid, rifampicin, ethambutol, para-aminosalicylic acid, streptomycin and kanamycin) have been documented by patch test to cause this type of dermatitis in certain individuals. Cross sensitization has been observed to contribute significantly to the allergic reactions noted from isoniazid, streptomycin, and kanamycin. Hyposensitization has also been discussed in this review.

Correlaçäo entre o preparo do cólon e infecçäo da incisäo cirúrgica / Correlation between colon preparation and surgical incision infection

Os autores, de acordo com os dados obtidos neste estudo, encontraram um efeito profilático da infecçäo incisional, em cirurgias prolongadas do cólon e reto, pela associaçäo da kenamicina ao preparo mecânico do cólon

Cross-sensitivity of common aminoglycoside antibiotics.

Guinea pigs were sensitized to neomycin (A, B, or C), paromomycin, gentamicin, kanamycin, streptomycin, and dihydrostreptomycin via intradermal or foot-pad injection with an adjuvant containing killed Mycobacterium butyricum or M tuberculosis H37Ra (Ra). These antibiotics produced greater cross-sensitization with an increase in the number of immunizations and chemical structural similarities. After repeated intradermal injections (adjuvant Ra) of neomycin, guinea pigs showed cross-sensitization to paromomycin, kanamycin, and streptomycin. A single intradermal injection of one of these antibiotics produced stronger reactions to the most closely related antibiotics, with no meaningful sensitization to the least-related allergens. Streptomycin-sensitized guinea pigs seldom showed a meaningful cross-sensitization to dihydrostreptomycin or the other antibiotics (except neomycin C); however, guinea pigs sensitized to dihydrostreptomycin or the other antibiotics often showed strong cross-sensitization to streptomycin.

Sensitization potentials and immunologic specificities of neomycins.

The use of sensitization indices for expressing allergenic skin reactions in guinea pigs is described. The method is convenient for comparing allergens and cross-reacting substances and permits the use of both irritating and nonirritating challenge concentrations of allergens. It also permits determination of both optimal reading time and challenge concentrations for each experiment. By this technique commercial neomycin complex, neamine (neomycin A), neomycin B, neomycin C, and streptomycin were found to be allergenic in guinea pigs via intradermal (id) and foot-pad (fp) immunizations. The immunizing emulsion consisted of an allergen and an adjuvant containing Mycobacterium butyricum (MB) or Mycobacterium tuberculosis H37Ra (Ra). The adjuvant MB was as effective as Ra by the id route, but inferior to Ra by the fp route. The cross-reactivity of neomycin C was generally greater than neomycin B in guinea pigs sensitized to neamine, neomycin B, neomycin C, or streptomycin. In guinea pigs sensitized to neomycin complex by repeated immunizations, neomycins A, B, and C were effective elicitors of skin reactions, whereas the N-acetylated derivatives of the components failed to cause reactions. This finding is interpreted to mean that the amino groups of the aminoglycosides are the coupling sites to host proteins in the processes of sensitization and elicitation of skin reactions in vivo.