Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antibiotics including sulfonamides, beta-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 µg/kg to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 µg/kg to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation fluid and those read fourteen hours after addition of the scintillation fluid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%.The Charm II tests were found to be 100% group specific, as none of the antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at Maximum Residue Limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antibiotics including sulfonamides, beta-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 µg/kg to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 µg/kg to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation fluid and those read fourteen hours after addition of the scintillation fluid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%.The Charm II tests were found to be 100% group specific, as none of antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study prove the specificity and precision of the Charm II assay in the detection of various antimicrobial residuals in fish. The results also demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at Maximum Residue Limits established in the EU guidelines, and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to European Commission Decision 2002/657/ EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antibiotics including sulfonamides, beta-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 µg/kg to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 µg/kg to 300 µg/kg and were 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation fluid and those read fourteen hours after addition of the scintillation fluid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranging from 0.02 to 0.1 was recorded.The Charm II tests were found to be 100% group specific, as none of antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. The results of this study prove the robustness, specificity, reliability and accuracy of the Charm II tests in the detection of various antimicrobial residuals in fish. Results also confirm the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species; and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

Recovery of 13CO2 and 14CO2 in Human Bicarbonate Studies: A Critical Review with Original Data

1. In order to establish biological and/or methodological explanations for the wide variability in recovery (50–100%) of labelled CO2 after administration of [13C]bicarbonate or [14C]bicarbonate, 34 human bicarbonate studies involving 480 subjects were analysed, and potential methodological issues were investigated in the laboratory. 2. Overall, continuous infusion studies reported a higher recovery than bolus studies (84 ± 11% versus 69 ± 12%; P < 0.001). No significant differences in recovery were found between 14C and 13C studies, children and adults, obese and lean subjects, or rest and exercise (steady state). Higher recoveries were found during feeding than during fasting (84 ± 8% versus 74 ± 7%; P < 0.001). Different methods used to analyse the results (0–10%) and different study protocols, which include differences in the duration of infusions and background drift in 13C enrichment (0–10%), contribute to the variability. 3. The laboratory studies suggest multiple sources of potential error, including loss of CO2 from the scintillation fluid (up to >30%, but only in 14C studies in which the scintillation fluid is not alkalized), diffusion of CO2 through syringes and tubing (0 to > 10%), non-linearity of CO2 analysers (up to 8%), inaccuracies in the measurement of bicarbonate concentrations (13C studies) or the strength of CO2-trapping agents (14C studies; 0–8%). 4. It is concluded that much of the variability in the recovery of labelled bicarbonate is likely to be attributable to methodological differences, and that attention to these will ensure better interpretation of metabolic studies that involve oxidation of carbon-labelled substrates.

Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique

A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.

Scintillation fluid shortens exposure times in autoradiography.

Premixed, commercially available scintillation fluids were used to reduce exposure time of tritium (3H) and iodine-125 (125I)-labeled whole cells, and of 3H-labeled Giemsa-banded chromosome preparations. Emulsion-coated slides were dipped into scintillator for no longer than 2 min and exposed in the dark at 4 degrees C. Maximal values for percentage of human diploid cell (WI-38) nuclei labeled with 3H-thymidine of moderate specific activity were obtained in 12 hr. Without scintillator the exposure time was 4 days. Exposure time for cells labeled with 125I-serum was reduced from over 90 days to 14 days. The shortened exposure time for banded chromosomes permitted successful prestaining with Giemsa, a sequence that is not possible without scintillator.