Lengthening the aptamer to hybridize with a stem-loop DNA assistant probe for the electrochemical detection of kanamycin with improved sensitivity.

By adding 6 thymines to lengthen the parent aptamer combined with the change of "on" and "off" induced by the target for an assistant stem-loop DNA probe (ASP-SLP-MB), a new folding-type electrochemical kanamycin (Kana) aptamer-engineering dual-probe-based sensor (sensor d) was developed. By purposefully reducing the background current and increasing the electron transfer efficiency of methylene blue (MB), the sensor obtained significantly enhanced detection sensitivity compared with non-aptamer-engineering one-probe-based sensor (sensor a). Such efficacy was validated by a big decrease from 530.6 to 210.2 nA for the background current signal and from 360 to 0.3 nM for the detection limit. In addition to the improved sensitivity, the sensor also exhibited good selectivity, anti-fouling detection performance, and potential quantitative analysis ability, showing a feasible potential practical analytical application in real-life complicated samples, for example, milk and serum. The released results prove that the aptamer-engineering method is effective in improving the analytical performance of folding-type sensors and provides a methodological guidance for the design and fabrication of other high-performance folding-type aptasensors. Graphical abstract.

Click chemistry inspired copper sulphide nanoparticle-based fluorescence assay of kanamycin using DNA aptamer.

A highly selective and sensitive fluorescence assay for kanamycin has been developed that depends on complementation of two splits of DNA aptamer. One DNA split was labeled with CuS nanoparticle and the other was decorated with biotin, which enabled coupling with streptavidin magnesphere paramagnetic particles (PMPs). Complementation of the two-aptamer splits happened only in the presence of kanamycin and the subsequent sandwich was separated via a magnet. The released Cu(II) was reduced to Cu(I) by sodium ascorbate and finally catalyzed the click reaction between fluorogenic 3-azido-7-hydroxycoumarin and propargyl alcohol to afford the corresponding fluorescent 1,4-disubstituted-1,2,3-triazole. The fluorescence signal produced (λex. = 365 nm, λem. = 470 nm) was dependent on kanamycin concentration. Fluorescence signal amplification was found to be in good linear relationship with the logarithm of kanamycin concentration in the range of 0.04-20 nM. Furthermore, the proposed assay showed a good reproducibility, high selectivity and low detection limits for kanamycin determination. In addition, the capability of the proposed method to detect kanamycin in biological samples with satisfactory results was demonstrated.

Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood.

The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clinically relevant (±20% for this application) accuracy, for example, commercial continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, we demonstrate a "dual-frequency" approach for achieving the calibration-free operation of electrochemical biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, we use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochemical aptamer-based (E-AB) biosensors as our test bed, we demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor.

Radiolabeling, quality control, and biological characterization of 177 Lu-labeled kanamycin.

Kanamycin is an antibiotic, isolated from Streptomyces kanamyceticus, which is used to treat serious bacterial infections. The fact that the present radioligand 99m Tc-kanamycin used for diagnosis is short-lived, raised a need to label and study kanamycin with one of the most important beta (ß) radiation emitting isotope 177 Lu. Labeling yield of 177 Lu-kanamycin was confirmed by different chromatography techniques such as paper chromatography, TLC, HPLC. Several experiments were performed to optimize labeling with changing reaction conditions such as pH, temperature, amount of ligand, and reaction time. In vitro stability analysis was performed incubation with human serum. Electrophoresis analysis was also conducted to determine the charge on 177 Lu-kanamycin. The biodistribution and scintigraphy were performed in normal mice and rabbit, respectively, at different time intervals of postinjection. 177 Lu-kanamycin was prepared with very high yield (~100%), with excellent stability in vivo and in vitro (>99% 6 hr postprep.), at pH 7. Maximum labeling was achieved at less reaction time (15 min), with maximum conjugation of the ligand (12.5 mg) with 177 Lu. Electrophoresis analysis showed net neutral charge. The radioligand showed rapid clearance from body in biodistribution and scintigraphy studies. The preparation 177 Lu-kanamycin could be used as a radio-pharmaceutical for infection imaging purpose, especially when transporting the radioligand to long-range distances.

Reduced Chance of Hearing Loss Associated with Therapeutic Drug Monitoring of Aminoglycosides in the Treatment of Multidrug-Resistant Tuberculosis.

Hearing loss and nephrotoxicity are associated with prolonged treatment duration and higher dosage of amikacin and kanamycin. In our tuberculosis center, we used therapeutic drug monitoring (TDM) targeting preset pharmacokinetic/pharmacodynamic (PK/PD) surrogate endpoints in an attempt to maintain efficacy while preventing (oto)toxicity. To evaluate this strategy, we retrospectively evaluated medical charts of tuberculosis (TB) patients treated with amikacin or kanamycin in the period from 2000 to 2012. Patients with culture-confirmed multiresistant or extensively drug-resistant tuberculosis (MDR/XDR-TB) receiving amikacin or kanamycin as part of their TB treatment for at least 3 days were eligible for inclusion in this retrospective study. Clinical data, including maximum concentration (Cmax), Cmin, and audiometry data, were extracted from the patients' medical charts. A total of 80 patients met the inclusion criteria. The mean weighted Cmax/MIC ratios obtained from 57 patients were 31.2 for amikacin and 12.3 for kanamycin. The extent of hearing loss was limited and correlated with the cumulative drug dose per kg of body weight during daily administration. At follow-up, 35 (67.3%) of all patients had successful outcome; there were no relapses. At a median dose of 6.5 mg/kg, a correlation was found between the dose per kg of body weight during daily dosing and the extent of hearing loss in dB at 8,000 Hz. These findings suggest that the efficacy at this lower dosage is maintained with limited toxicity. A randomized controlled trial should provide final proof of the safety and efficacy of TDM-guided use of aminoglycosides in MDR-TB treatment.

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.

Quantification of amikacin and kanamycin in serum using a simple and validated LC-MS/MS method.

BACKGROUND: Amikacin and kanamycin are frequently used in the treatment of multidrug-resistant TB. The current commercially available immunoassay is unable to analyze kanamycin and trough levels of amikacin. The objective was therefore to develop a LC-MS/MS method for the quantification of amikacin and kanamycin in human serum. MATERIALS & METHODS: Using apramycin as internal standard, selectivity, accuracy, precision, recovery, matrix effects and stability were evaluated. RESULTS: The presented LC-MS/MS method meets the recommendations of the US FDA with a low LLOQ of 250 ng/ml for amikacin and 100 ng/ml for kanamycin. No statistical significant difference was found between the LC-MS/MS method and the immunoassay of amikacin (Architect(®) assay, p = 0.501). CONCLUSION: The low LLOQ of amikacin and the ability to analyze kanamycin makes the LC-MS/MS method the preferred method for analyzing these aminoglycosides.

An ultrasensitive homogeneous aptasensor for kanamycin based on upconversion fluorescence resonance energy transfer.

We developed an ultrasensitive fluorescence resonance energy transfer (FRET) aptasensor for kanamycin detection, using upconversion nanoparticles (UCNPs) as the energy donor and graphene as the energy acceptor. Oleic acid modified upconversion nanoparticles were synthesized through a hydrothermal process followed by a ligand exchange with hexanedioic acid. The kanamycin aptamer (5'-NH2-AGATGGGGGTTGAGGCTAAGCCGA-3') was tagged to UCNPs through an EDC-NHS protocol. The π-π stacking interaction between the aptamer and graphene brought UCNPs and graphene in close proximity and hence initiated the FRET process resulting in quenching of UCNPs fluorescence. The addition of kanamycin to the UCNPs-aptamer-graphene complex caused the fluorescence recovery because of the blocking of the energy transfer, which was induced by the conformation change of aptamer into a hairpin structure. A linear calibration was obtained between the fluorescence intensity and the logarithm of kanamycin concentration in the range from 0.01 nM to 3 nM in aqueous buffer solution, with a detection limit of 9 pM. The aptasensor was also applicable in diluted human serum sample with a linear range from 0.03 nM to 3 nM and a detection limit of 18 pM. The aptasensor showed good specificity towards kanamycin without being disturbed by other antibiotics. The ultrahigh sensitivity and pronounced robustness in complicated sample matrix suggested promising prospect of the aptasensor in practical applications.

Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis.

Little is known about plasma drug concentrations relative to quantitative susceptibility in patients with multidrug-resistant tuberculosis (MDR-TB). We previously described a TB drug activity (TDA) assay that determines the ratio of the time to detection of plasma-cocultured Mycobacterium tuberculosis versus control growth in a Bactec MGIT system. Here, we assess the activity of individual drugs in a typical MDR-TB regimen using the TDA assay. We also examined the relationship of the TDA to the drug concentration at 2 h (C2) and the MICs among adults on a MDR-TB regimen in Tanzania. These parameters were also compared to the treatment outcome of sputum culture conversion. Individually, moxifloxacin yielded superior TDA results versus ofloxacin, and only moxifloxacin and amikacin yielded TDAs equivalent to a -2-log killing. In the 25 patients enrolled on a regimen of kanamycin, levofloxacin, ethionamide, pyrazinamide, and cycloserine, the C2 values were found to be below the expected range for levofloxacin in 13 (52%) and kanamycin in 10 (40%). Three subjects with the lowest TDA result (<1.5, a finding indicative of poor killing) had significantly lower kanamycin C2/MIC ratios than subjects with a TDA of ≥1.5 (9.8 ± 8.7 versus 27.0 ± 19.1; P = 0.04). The mean TDAs were 2.52 ± 0.76 in subjects converting to negative in ≤2 months and 1.88 ± 0.57 in subjects converting to negative in >2 months (P = 0.08). In Tanzania, MDR-TB drug concentrations were frequently low, and a wide concentration/MIC range was observed that affected plasma drug activity ex vivo. An opportunity exists for pharmacokinetic optimization in current MDR-TB regimens, which may improve treatment response.

Real-time, aptamer-based tracking of circulating therapeutic agents in living animals.

A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients' health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system's versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at subminute temporal resolution. We show that MEDIC can also obtain pharmacokinetic parameters for individual animals in real time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe that MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.

Study of drug transport between the blood and lymph in the predominant direction.

Our method for evaluating the time course and intensity of antibiotics and other drugs transport in the predominant direction between the blood and lymph in humans promotes a more objective evaluation of drug circulation mechanisms, which is essential for determining the time of their repeated administration and route of administration. Calculation of the lymph/blood difference coefficient, based on parallel repeated measurements of the drug concentration in the lymph and blood, and of the lymph/blood coefficient provides complete data on the direction and time course of drug transport between the lymph and blood in the predominant direction.

Determination of neomycin and bacitracin in human or rabbit serum by HPLC-MS/MS.

The method for the simultaneous determination of neomycin and bacitracin in human or rabbit serum was developed by using ion pairing reversed phase chromatography and tandem mass spectrometry (MS/MS) detection with electrospray (ESI) in positive mode. Both substances elute under these conditions at the same time and also kanamycin as internal standard elutes almost at the same time. The sample preparation was simple-only using 0.1 mL serum by protein precipitation with acetonitrile. Neomycin and bacitracin were detected as two-fold charged ions as well as the internal standard. The calibration range of these quite difficult detectable substances was 0.2-50 microg/mL of serum. The method was validated for both human or rabbit serum. The inter batch precision of quality control samples in human serum for neomycin ranged from 4.46% to 8.99% and for bacitracin from 6.85% to 11.17%. The inter batch accuracy for neomycin ranged from 98.7% to 100.7% and for bacitracin from 99.2% to 103.0%. At lower limit of quantitation (LLOQ) level of 0.2 microg/mL inter batch precision in human serum for neomycin was 12.05% and for bacitracin 11.91%, whereas accuracies were 99.9% for neomycin and 102.7% for bacitracin. Bench top stability in human or rabbit serum was given over three freeze thaw cycles and 4h at room temperature. The method can be considered to be specific and recoveries for sample preparation were high.

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

A simple and sensitive HPLC method has been developed for trace determination of kanamycin A by derivatization. Plasma proteins are precipitated by acetonitrile and chemical derivatization is performed on the supernatant containing kanamycin A with 1-naphthyl isothiocyanate in pyridine at 70 degrees C. After the derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess of derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Lichrocart Purospher STAR RP-18e column and water/methanol (33:67, v/v) as a mobile phase (detection at 230 nm). Optimization conditions for the derivatization of kanamycin A were investigated by HPLC. The linear range for the quantitation of kanamycin A in spiked plasma was over 1.2-40 microg/mL; the detection limit (signal to noise ratio = 3; injection volume, 10 microL) was about 0.3 microg/mL. The relative standard deviation was less than 2.9% for intra-day assay (n = 6) and inter-day assay (n = 6) and relative recoveries were found to be greater than 98%. Preliminary application of the method for monitoring kanamycin A in humans upon intramuscular injection of the injection product demonstrated the usefulness of the assay for clinical studies. The proposed method can also be used to analyze the compound in pharmaceutical formulations.

Determination of tobramycin in human serum by capillary electrophoresis with contactless conductivity detection.

A study on the determination of the antibiotic tobramycin by CE with capacitively coupled contactless conductivity detection is presented. This method enabled the direct quantification of the non-UV-absorbing species without incurring the disadvantages of the indirect approaches which would be needed for optical detection. The separation of tobramycin from inorganic cations present in serum samples was achieved by optimizing the composition of the acetic acid buffer. Field-amplified sample stacking was employed to enhance the sensitivity of the method and a detection limit of 50 microg/L (S/N = 3) was reached. The RSDs obtained for migration time and peak area using kanamycin B as internal standard were typically 0.12 and 4%, respectively. The newly developed method was validated by measuring the concentration of tobramycin in serum standards containing typical therapeutic concentrations of 2 and 10 mg/L. The recoveries were 96 and 97% for the two concentrations, respectively.

Tanshinone (Salviae miltiorrhizae extract) preparations attenuate aminoglycoside-induced free radical formation in vitro and ototoxicity in vivo.

Antioxidant therapy protects against aminoglycoside-induced ototoxicity in animal models. A clinically suitable antioxidant must not affect the therapeutic efficacy of aminoglycosides or exhibit any side effects of its own. In addition, the treatment should be inexpensive and convenient in order to be implemented in developing countries where the use of aminoglycosides is most common. Standardized Salviae miltiorrhizae extracts (Danshen) are used clinically in China and contain diterpene quinones and phenolic acids with antioxidant properties. We combined in vitro and in vivo approaches to investigate the effect of a clinically approved injectable Danshen solution on aminoglycoside-induced free radical generation and ototoxicity. In vitro, Danshen inhibited gentamicin-dependent lipid peroxidation (formation of conjugated dienes from arachidonic acid), as well as the gentamicin-catalyzed formation of superoxide (in a lucigenin-based chemiluminescence assay) and hydroxyl radicals (oxidation of N,N-dimethyl-p-nitrosoaniline). Danshen extracts were then administered to adult CBA mice receiving concurrent treatment with kanamycin (700 mg/kg of body weight twice daily for 15 days). Auditory threshold shifts induced by kanamycin (approximately 50 dB) were significantly attenuated. Danshen did not reduce the levels in serum or antibacterial efficacy of kanamycin. These results suggest that herbal medications may be a significantly underexplored source of antidotes for aminoglycoside ototoxicity. Such traditional medicines are widely used in many developing countries and could become an easily accepted and inexpensive protective therapy.

Determination of kanamycin in serum by solid-phase extraction, pre-capillary derivatization and capillary electrophoresis.

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat.

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Overexpression of copper/zinc-superoxide dismutase protects from kanamycin-induced hearing loss.

The participation of reactive oxygen species in aminoglycoside-induced ototoxicity has been deduced from observations that aminoglycoside-iron complexes catalyze the formation of superoxide radicals in vitro and that antioxidants attenuate ototoxicity in vivo. We therefore hypothesized that overexpression of Cu/Zn-superoxide dismutase (h-SOD1) should protect transgenic mice from ototoxicity. Immunocytochemistry confirmed expression of h-SOD1 in inner ear tissues of transgenic C57BL/6-TgN[SOD1]3Cje mice. Transgenic and nontransgenic littermates received kanamycin (400 mg/kg body weight/day) for 10 days beginning on day 10 after birth. Auditory thresholds were tested by evoked auditory brain stem responses at 1 month after birth. In nontransgenic animals, the threshold in the kanamycin-treated group was 45-50 dB higher than in saline-injected controls. In the transgenic group, kanamycin increased the threshold by only 15 dB over the respective controls. The effects were similar at 12 and 24 kHz. The protection by overexpression of superoxide dismutase supports the hypothesis that oxidant stress plays a significant role in aminoglycoside-induced ototoxicity. The results also suggest transgenic animals as suitable models to investigate the underlying mechanisms and possible strategies for prevention.

[Pharmacokinetics of kanamycin after subcutaneous and intravenous administration in dogs]. / Pharmakokinetik von Kanamycin nach subkutaner und intravenöser Applikation beim Hund.

Six beagle dogs were treated with kanamycin subcutaneously or intravenously in a dosage of 5 mg/kg. The plasma kanamycin concentration was measured over 24 hours by high pressure liquid chromatography with UV detection after derivatization and solid phase extraction. After subcutaneous application, kanamycin was absorbed quickly, and maximum plasma levels of 18.9 micrograms/ml in average after ca. 1 hour were measured. With complete systemic availability, the minimal inhibitory concentration of 4 micrograms/ml was maintained for 4 hours. After subcutaneous administration, kanamycin was terminally eliminated with a mean half life period of 2 hours.

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.