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• Comparison of NI susceptibility enzyme inhibition assays

The IC50 value generated in the enzyme inhibition assay is affected by a number of parameters.  Firstly there is a significant difference for influenza B values in the chemiluminescent (CL) compared to the fluorescent assay (FL).  The values for influenza B in the CL assay are within the low nM range for both drugs, values which are only marginally higher compared to those observed for the influenza A H1N1 and H3N2 NAs (Wetherall et al 2003. J Clin Micro; McKimm-Breschkin et al 2003. Antimicrob Agents Chemother).  In the FL assay, however, the influenza B IC50 values are around 10-fold or more higher for oseltamivir compared to zanamivir, and hence are at least an order of magnitude higher than the IC50 values for oseltamivir for H1N1 and H3N2 NAs.  

There are also differences seen with different mutant NAs between the two assays, for example in the FL assay the IC50 of the E119V mutant NA for oseltamivir is usually several hundred nM.  In contrast, the IC50 for the E119V mutant NA for oseltamivir in the CL assay is only around 10 nM.   In addition to differences between the two types of assay, the buffer, the pH and the substrate concentration of the reaction mix can also affect both the enzyme activity, and the IC50.  The table below illustrates the difference in IC50 values according to fluorescent (FL) and chemiluminescent (CL) assay methodology used.

NISN is currently aiming to develop an assay methodology which maximizes enzyme activity, hence enabling lower amounts of virus to be detected, as well as enabling identification of known resistant phenotypes.