| Assay Method Information | |
| | Neuraminidase Inhibition Assay |
| Description: | Purified hPIV-3 or hPIV-1, inhibitors and MUN were prepared and diluted in NA Reaction Buffer [NaOAc 50 mM, CaCI2 5 mM, pH 4.6 (hPIV-3) or 5.0 (hPIV-1)]. Neuraminidase assay, employing different hPIV-3 or hPIV-1 dilutions, were initially measured to determine the lowest virus concentration to be used in the assays. The neuraminidase assays were performed with enough purified virus to obtain a maximal fluorescence signal at least 5 times higher than the background for the experiment to be considered statistically significant. Neuraminidase inhibition (N1) assays were done in triplicate in 384 well black plate. For each concentration tested, 2 μL of purified hPIV and 4 μL of 2.5× inhibitor solution (1× final) was added to each well. The plate was kept at room temperature for 20 min before 4 μL of 5 mM 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUN) (2 mM final) was added to each well and then the plate incubated at 37° C. for 30 min with agitation (1100 rpm). The enzymatic reaction was stopped by the addition of 50 μI of glycine buffer (glycine 0.25 M, pH 10.4) to each well. A negative control was included by the addition of MUN to virus and then the enzymatic reaction stopped at t=0 min. Relative fluorescence (RF) was measured with a Tecan Infinite M200 Pro. Data were processed by background subtraction (negative control RF) and then analysed with GraphPadPrism to calculate IC50 values (nonlinear regression (curve fit), Dose-response - inhibition, 3 or 4 parameter logistic). The concentration of inhibitor that reduced neuraminidase activity (relative fluorescence) by 50% compared to those of a non-treated virus suspension was considered to be the neuraminidase IC50 value. |
| Affinity data for this assay | |
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