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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.

MUNANA, the substrate compound in the fluorescent assay, was previously only available from the laboratory chemical supply company Sigma-Aldrich. However, MUNANA can now be obtained from various other companies, many of which are providing the compound at a significantly lower cost. The World Health Organization Global Influenza Surveillance and Response System Expert Working Group on Surveillance of Influenza Antiviral Susceptibility tested MUNANA obtained from two different companies - Biosynth AG and Sequoia Research Products Ltd - and compared their performance to MUNANA from Sigma-Aldrich. MUNANA from Biosynth and Sequoia performed well in NA inhibition assays with either superior or comparable activity to that of the Sigma-Aldrich MUNANA. Although analytical performance is comparable, the price of MUNANA from these companies can be considerably lower; therefore researchers are encouraged to investigate some of these alternative suppliers -  Click here for alternative sources MUNANA.
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.