| Assay Method Information | |
| | Neuraminidase Activity Assay |
| Description: | Practically in a microtitre plate, 90 μl of 33 mM MES pH 6.5 containing 4 mM CaCl2 were dispensed in each well, except for the first well of each lane. In the first well of each vertical lane, 18 μl of 0.01 mM oseltamivir were mixed with 162 μl of 33 mM MES pH 6.5 containing 4 mM CaCl2. The solution was thoroughly mixed to obtain a 1-μM oseltamivir carboxylate solution. For each assay, a 90-μl aliquot of this solution was withdrawn from the first well and added to the second well in the same vertical lane obtaining a 2-fold diluted solution of oseltamivir. The 2-fold dilution process was continued up to the seventh well, while the eighth well used as blank (no inhibitor was added). At the end of the dilution process, each well contained 90 μl of solution. To each well, 25 μl of enzyme stock solution were added. After an incubation time of 2 h, 25 μl of MUNANA were added to each well, the final concentrations of oseltamivir carboxylate spanned the range 10-643 nM for N1 assay. After 30 min of incubation, 50 μl of stop solution were added to the first vertical lane, and the fluorescence was recorded. After an additional 30 min (60 min total), the stop solution was added to the second vertical lane and the fluorescence recorded. After additional 30 min (90 min total), the stop solution was added to the third vertical lane and the fluorescence recorded and so on. The experiment was performed in duplicate. For the determination of oseltamivir carboxylate Ki against N3, the same procedure described for N1 was used, except for the concentration of oseltamivir carboxylate stock solution which was 0.1 μM. |
| Affinity data for this assay | |
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