Table 1.
Calculation of the spectroscopic correction factor.
Figure 1.
Standard curve of 4-methylumbelliferone (4-MU) fluorescence.
Fluorescence intensity was measured using a Synergy 2 multimode microplate reader at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Relative fluorescence units (RFU) obtained at low 4-MU concentrations (0–2.0 µM) are shown in the insert. Each data point represents the mean ± standard deviation (SD) of 10 independent measurements.
Figure 2.
Influenza A/CA/04/09 (H1N1pdm09) virus dilution selection for determination of NA enzyme kinetics parameters.
Two-fold dilutions of A/CA/04/09 (H1N1pdm09) virus were prepared in enzyme buffer. The graph shows virus dilutions (1∶16 to 1∶1024) that generated linearly increasing amounts of 4-MU over the reaction time (R2>0.99). Fluorescence intensity was recorded every 60 s for 60 min at 37°C with the MUNANA substrate at a final concentration of 100 µM. Blank signal value determined from reactions without virus was subtracted from RFU generated in the NA enzymatic reactions. The virus dilution of 1∶64 was selected in this case for further studies on the basis of linearity of the curve (dotted line), signal-to-noise ratio ≥10, and conversion of ≤15% of the total amount of MUNANA after 60 min.
Figure 3.
Possible kinetic profiles of NA enzyme reactions at different MUNANA substrate concentrations.
Fluorescence intensity was recorded every 60 s for 60 min at 37°C with the MUNANA substrate at the final concentration of 0.1–1000 µM. (A) Profile consistent with steady-state conditions during the time of the reaction. (B) Dataset with initial lag phase (shown in red parentheses) and (C) datasets with lag (shown in red parentheses) and saturated phases (shown in blue parentheses) indicate suboptimal assay conditions and a requirement for assay optimization.
Figure 4.
Inner filter effect (IFE) from light absorption at the excitation and emission wavelengths of the 4-MU product.
(A) Absorbance spectra of MUNANA and 4-MU at 0.01 mg/mL and 0.1 mg/mL concentrations in enzyme buffer. Optical density was measured using Synergy 2 multi-mode microplate reader in a UV-transparent 96-well plate. (B) Absorbance spectra of MUNANA and 4-MU at 0.1 mg/mL concentration. (C) 4-MU fluorescence measured in the presence of different concentrations of MUNANA (15–2000 µM) shows similar impact of MUNANA-associated spectroscopic interference across 4-MU concentrations of 10–80 µM.
Figure 5.
NA enzyme kinetics curves with and without spectroscopic corrections.
(A) Data for A/PR/8/34 (H1N1) influenza virus; (B) A/CA/04/09 (H1N1pdm09) influenza virus and (C) B/WIS/01/10 influenza virus. Each data point represents the mean of 4 (A/CA/04/09 and B/WIS/01/10 viruses) or 7 (A/PR/8/34 virus) independent measurements. Dotted lines represent data without spectroscopic correction and solid lines represent data fitting to a hyperbolic equation after correction.
Table 2.
Neuraminidase enzyme kinetics parameters of influenza viruses.
Figure 6.
Flowchart for the determination of NA enzyme kinetic parameters using whole influenza virus preparations.