The viruses were diluted in Opti-MEM (Gibco) and 45 ml of titrated virus was added to 45 ml of Opti-MEM into a 96-well plate and incubated for 1 h at 37 C. MCDK-SIAT1-PB1 cells were trypsinized, washed once with PBS and resuspended in Opti-MEM. Thirty ml of MDCK-SIAT1-PB1 cells at a concentration of 1 3 10 6 cells ml -1 were added to each well containing the diluted virus. Twenty-five ml of virus-cell mixture was added to each 384 well in quadruplicate and incubated for 20-24 h at 37 C with 5% CO 2 . Next day, mAbs were diluted in the assay buffer (NA-Fluor Influenza Neuraminidase Assay Kit; Thermo Fisher Scientific) at a starting concentration of 50 mg ml -1 . Culture supernatant of the 384-well plate was discarded and replenished with 25 ml diluted mAbs in quadruplicate. The plates were incubated for 1 h at 37 C with 5% CO 2 , and 25 mL of assay substrate (NA-Fluor Influenza Neuraminidase Assay Kit; Thermo Fisher Scientific) was added to each well and incubated for another 1 h at 37 C with 5% CO 2 . Reaction was then stopped by adding 50 ml of stop solution (NA-Fluor Influenza Neuraminidase Assay Kit; Thermo Fisher Scientific), and the plates were read using an excitation wavelength range of 350 nm to 365 nm and an emission wavelength range of 440 nm to 460 nm. Control wells without the virus infection were used for background subtraction.
Na fluor influenza neuraminidase assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NA-Fluor™ Influenza Neuraminidase Assay Kit is a fluorescence-based assay used for the quantitative measurement of influenza neuraminidase activity.
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Lab products found in correlation
Fluorescence spectrometer, by Promega (4 mentions)
Fluorescence spectrophotometer, by Promega (2 mentions)
Munana, by Merck Group (2 mentions)
Oseltamivir carboxylate, by Aobious (2 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Zanamivir, by GlaxoSmithKline (1 mentions)
Oseltamivir carboxylate oseltamivir, by Roche (1 mentions)
Glomax explorer system, by Promega (1 mentions)
Prism 5, by GraphPad (1 mentions)
Oseltamivir carboxylate, by Roche (1 mentions)
Luminescence plate reader, by Promega (1 mentions)
Absolute ethanol, by Merck Group (1 mentions)
Fetuin from fetal calf serum, by Merck Group (1 mentions)
α2 3 sialyllactose, by Merck Group (1 mentions)
α2 6 sialyllactose, by Merck Group (1 mentions)
Spectramax m5e microplate reader, by Molecular Devices (1 mentions)
4 methylumbelliferone 4 mu, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
33 protocols using na fluor influenza neuraminidase assay kit
1
Influenza Neuraminidase Inhibition Assay
2
Influenza Neuraminidase Activity Assay
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The neuraminidase (NA) activities of the viruses were measured with an NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems). Briefly, serial dilutions of the viruses in 96-well black-wall clear-bottom imaging plates were incubated with the fluorescent NA substrate (4-methylumbelliferyl-N-acetylneuraminic acid) at 37°C for 1 hour prior to assay termination by addition of 100 μl of the NA-Fluor stop solution. The fluorescence intensities were measured on an EnVision plate reader (PerkinElmer). The NA activities as a function of the fluorescence intensities in relative fluorescence unit (RFU) were plotted on the y-axis versus the log10 virus dilutions on the x-axis. The data were fit with a nonlinear regression dose-response curve to determine the optimal dilution for each virus.
Virus dilutions with equal NA activities were used in the oseltamivir acid inhibition assay. Each virus preparation in duplicate was incubated with varying concentrations of oseltamivir acid for 30 minutes at 37°C. The fluorescent NA substrate was then added and incubated for 1 hour at 37°C prior to assay termination. The fluorescence intensities were measured as above. The NA activities were normalized for each virus and the percentages of NA activities were plotted on the y-axis versus the log10 oseltamivir acid concentrations on the x-axis.
Virus dilutions with equal NA activities were used in the oseltamivir acid inhibition assay. Each virus preparation in duplicate was incubated with varying concentrations of oseltamivir acid for 30 minutes at 37°C. The fluorescent NA substrate was then added and incubated for 1 hour at 37°C prior to assay termination. The fluorescence intensities were measured as above. The NA activities were normalized for each virus and the percentages of NA activities were plotted on the y-axis versus the log10 oseltamivir acid concentrations on the x-axis.
3
Neuraminidase Inhibition Assay for Influenza
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The NA inhibitors oseltamivir carboxylate (oseltamivir; Hoffman-La Roche) and zanamivir (GlaxoSmithKline) were prepared in sterile distilled water and stored in aliquots at − 30 °C until use.
Inhibition of NA enzyme activity by the 2 NA inhibitors was assessed in the fluorescence-based NA inhibition assay using the NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems, ThermoFisher) as previously described [15 (link)]. IC50 values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad Prism 5 software. The IC50 values reported are the means (±SD) of IC50 values measured for at least 2 independent tests with 2 duplicates for each sample. Interpretation of IC50 values (obtained by comparing the test virus with the drug-sensitive reference virus) was performed using the WHO AVWG criteria for influenza A viruses: a < 10-fold increase in IC50 represents normal inhibition, a 10~100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.
Inhibition of NA enzyme activity by the 2 NA inhibitors was assessed in the fluorescence-based NA inhibition assay using the NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems, ThermoFisher) as previously described [15 (link)]. IC50 values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad Prism 5 software. The IC50 values reported are the means (±SD) of IC50 values measured for at least 2 independent tests with 2 duplicates for each sample. Interpretation of IC50 values (obtained by comparing the test virus with the drug-sensitive reference virus) was performed using the WHO AVWG criteria for influenza A viruses: a < 10-fold increase in IC50 represents normal inhibition, a 10~100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.
4
Influenza Neuraminidase Inhibition Assay
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Oseltamivir and zanamivir inhibition of the NA activity of virus isolates was assessed using the NA-Fluor Influenza Neuraminidase Assay Kit from Applied Biosystems (Life technologies). The reference virus A/California/07/2009(H1N1pdm09) was used as wild type control. The mean 50% inhibitory concentration (IC) was calculated according to the kit protocol and as the fold-change to the wild-type control virus. World Health Organization (WHO) criteria for determination of inhibition by oseltamivir and zanamivir were used to evaluate the level of antiviral resistance [20 (link)]. Normal inhibition (NI) is < 10 IC50 fold change compared with wild-type virus, reduced inhibition (RI) 10–100, and highly reduced (HR) > 100.
5
Influenza Neuraminidase Inhibition Assay
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NA inhibition assays were conducted using the NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Serially diluted TSN in assay buffer was added to black 96-well plates at 25 µl/well. Then, 25 µl A/PR/8/34 or H3N2 was added to each well, and the mixture was incubated for 30 min at 37°C, followed by 50 µl of 200 µM NA-Fluor Substrate. After 1 h incubation at 37°C, 100 µl NA-Fluor stop solution was added to terminate the reaction and was monitored by GloMax Explorer System (Promega, Madison, WI).
6
Neuraminidase Inhibition Assay Protocol
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Inhibition of NA enzyme activity of the 4 NA inhibitors was assessed in the fluorescence-based NA inhibition assay, using the NA-Fluor Influenza Neuraminidase Assay kit (Applied Biosystems, ThermoFisher) as previously described47 (link). IC50 values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad prism5 software. Interpretation of IC50 was performed using the WHO Antiviral Working Group (WHO-AVWG) criteria47 (link)–49 : the testing virus was compared with the drug-sensitive reference virus, for influenza A viruses, a < tenfold increase in IC50 represents normal inhibition, and a ten to 100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.
7
Fluorescence-Based Neuraminidase Inhibition Assay
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A fluorescence-based neuraminidase inhibition assay was conducted using the NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems), according to the manufacturer’s instructions. The susceptibility of influenza viruses to NAI was characterized using oseltamivir, zanamivir, and peramivir at concentrations that inhibited the NA activity by 50% (IC50) as described previously [14 (link)]. As defined by the WHO, influenza A viruses with normal, reduced, and highly reduced NA activity inhibition exhibit a < 10-fold, 10- to 100-fold, and ≥ 100-fold increase in IC50, respectively; the last are considered clinically resistant [15 ].
8
Neuraminidase Inhibitor Susceptibility Assay
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Cell-based assays are not suitable for monitoring the susceptibility of influenza viruses to NA inhibitors because amino acid substitutions in the hemagglutinin protein can affect the NA inhibitor susceptibility in the cell-based assays (Barnett et al., 2000 (link); Tisdale, 2000 (link)). Therefore, the susceptibilities of the viruses to NA inhibitors were determined by using a fluorescence-based NA inhibition assay with the NA-Fluor influenza neuraminidase assay kit (Applied Biosystems). The results are expressed as the 50% inhibitory concentration (IC50). The IC50 values were calculated by using GraphPad Prism (GraphPad Software). To interpret NA inhibitor susceptibility, we used the World Health Organization (WHO) criteria, which are based on the fold-change in IC50 compared to the median for viruses from the same type/subtype/lineage showing normal inhibition (NI) (WHO, 2012 (link)). Reduced inhibition (RI) is defined as a 10- to 100-fold increase in IC50 for influenza A viruses, or a 5- to 50-fold increase in IC50 for influenza B viruses. Viruses showing highly reduced inhibition (HRI) are influenza A viruses with >100-fold increase in IC50 or influenza B viruses with >50-fold increase in IC50.
9
Oseltamivir Susceptibility of H1N1 Virus
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The susceptibility of A/Tianjin-baodi/1606/2018(H1N1) to the NA inhibitor oseltamivir carboxylate (Roche Diagnostics GmbH) was evaluated using an NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems). Oseltamivir-resistant virus (A/Texas/23/2012(H1)Y275) and oseltamivir-sensitive virus (A/Texas/23/2012(H1)H275), provided by CNIC, were used as controls. The half maximal inhibitory concentration (IC50), which represented the concentration of oseltamivir that could inhibit 50% of NA activity, was used to evaluate the antiviral susceptibility of the virus. Viruses are considered to show normal inhibition (NI) if IC50 is increased no more than 10-fold compared to the oseltamivir-sensitive control virus, reduced inhibition (RI) if the IC50 is increased 10–100-fold and highly reduced inhibition (HRI) if IC50 is increased >100-fold (see the WHO web site https://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/nai_overview/en/ ).
10
Quantifying Viral Neuraminidase Activity
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The NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA, USA) was used to measure the NA activity according to the manufacturer's instructions. Briefly, VB was combined with an assay buffer in a 96-well plate at 0–100 μmol/L for H1N1, H3N2, and influenza B viruses. Next, H1N1, H3N2, or influenza B were added to the VB-containing wells and incubated at 37 °C. Oseltamivir was used as a positive control in the assay. After 30 min, NA-Fluor substrate was added to each well and incubated for extra 2 h followed by measurement (excitation: 365 nm; emission: 415–445 nm) with a fluorescence spectrophotometer (Promega, Madison, WI, USA).
For the NA-XTD influenza neuraminidase assay, VB or oseltamivir carboxylate were added to the assay buffer in 96-well plates at 0–100 μmol/L for oseltamivir-resistant A/PR8/34. Subsequently, oseltamivir-resistant A/PR8/34 in assay buffer was added to VB or oseltamivir carboxylate-containing wells and incubated at 37 °C. Oseltamivir carboxylate was considered as the positive control in the assay. After 20 min, NA-XTD substrate was added to each well and incubated for an additional 30 min. Next, NA-XTD accelerator was added to each well, followed by recording the luminescence using a luminescence plate reader (Promega, Madison, WI, USA).
For the NA-XTD influenza neuraminidase assay, VB or oseltamivir carboxylate were added to the assay buffer in 96-well plates at 0–100 μmol/L for oseltamivir-resistant A/PR8/34. Subsequently, oseltamivir-resistant A/PR8/34 in assay buffer was added to VB or oseltamivir carboxylate-containing wells and incubated at 37 °C. Oseltamivir carboxylate was considered as the positive control in the assay. After 20 min, NA-XTD substrate was added to each well and incubated for an additional 30 min. Next, NA-XTD accelerator was added to each well, followed by recording the luminescence using a luminescence plate reader (Promega, Madison, WI, USA).
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