isirv Antiviral Group
Neuraminidase Inhibition Assay (IC50)
The simplest and clearest method to measure NA inhibitor susceptibility is by enzyme inhibition assay. There are two assays that may be used, both of which use small defined substrates.
The most widely used is a fluorescence assay which uses the substrate 20-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA). Cleavage of MUNANA by neuraminidase releases the fluorescent product methylumbelliferone. The amount of fluorescence therefore directly relates to the amount of enzyme activity. There are several SOPs available for assays using this substrate, which is also now available in a kit format. NA-FLUOR® Influenza NAI Resistance Detection Kit.
The second assay uses a chemiluminescent substrate, 20-(4-NA-Star)-a-D-N-acetylneuraminic acid (NA-Star), which is sold as part of a kit. NA-Star® Influenza NAI Resistance Detection Kit
A second chemiluminescent assay kit is now available with a modified substrate, which gives a long-lasting chemiluminescent signal, offering improved ease of use. NA-XTD® Influenza NAI Resistance Detection Kit
Any isolate suspected of showing reduced susceptibility in the NA inhibition assay should be further characterised by sequencing the NA gene to better define its NAI susceptibility. Resistance cannot be determined by sequencing the NA gene alone as the mechanisms of resistance are not yet fully understood and there may be resistance mutations which have not yet been identified.
- Kits and Reagents for IC50 testing (provided by the HPA, London)
- IC50 analysis protocol (provided by the HPA, London)
If you are using the IC50 analysis protocol provided above, the data can be analysed using commercially available software such as Graphpad Prism or Grafit. If this software is not available the Excel spreadsheets provided below can be used to analyse your data.