| Assay Method Information | |
| | Neuraminidase Inhibition Assay |
| Description: | The 50% inhibitory concentration (IC50) determination was carried out by using a 96-well microtitre plate as an array of reaction vessels. In the first well,a 0.1-mM solution of oseltamivir carboxylate was prepared by mixing 90 μl of 33 mM MES pH 6.5 containing 4 mM CaCl2 and 10 μl of a 1-mM oseltamivir carboxylate stock solution. Starting from the solution contained in the first well, an in-plate 10-fold serial dilution was performed using 33 mM MES pH 6.5 containing 4 mM CaCl2 as dilution buffer obtaining a 7 points dilution curve, the last well in the vertical lane was used to obtain a negative control (no oseltamivir was added). At the end of the dilution process, each well contained 90 μl of solution. To each well, 25 μl of the enzyme stock solution were mixed with the oseltamivir solution and incubated for 2 h at 37°C. After the incubation time, 25 μl of 20 μM MUNANA were added. In the reaction mixtures, the final concentration of oseltamivir carboxylate spanned in the ranges 0.064 nM-64.2 μM. After incubating the plate for 5 h at 37°C, 50 μl of stop solution (0.1 M Glycine, pH 10.7 containing 25% ethanol) were added. The fluorescence of the solutions contained in each well was read with the Victor3 multi-label counter. For the determination of the of oseltamivir carboxylate against neuraminidase N3, the same procedure described above was used, except for the concentrationof the oseltamivir carboxylate stock solution which was 10 μM. In the reaction mixtures, the final concentration of oseltamivir carboxylate spanned in the ranges 0.64 pM-642 nM. |
| Affinity data for this assay | |
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