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  • IC50 protocols
 

Whilst NISN does not currently endorse a specific methodology for undertaking IC50 analysis the following protocol may be beneficial to laboratories that are starting up.  This protocol been used to prepare the NISN Virus Reference Panel and will thus aid standardisation and validation of results if used alongside the panel of reference strains.

Acrobat document MUNANA assay protocol (Provided by Dr Aeron Hurt)

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.

The table below summarises the IC50 enzyme inhibition assay methodologies currently used by members according to their local requirements, and provides the protocols to download.  If you have any queries regarding these protocols please contact NISN.

Table: IC50 protocols used by NISN members, with variations noted where applicable


  Provided by  Published method     Substrate     Variation from published methodology  Protocol
  Dr Aeron Hurt  Ref 1
(FA2)		  MUNANA  Incubation for 1hr rather than 2hr
Document Acrobat
  Dr Elena Govorkova  Ref 2  MUNANA  As published method
Document Acrobat
  Dr Jennifer McKimm-Breschkin     Ref 3  MUNANA  As published method
Document Acrobat
  Prof Maria Zambon  Ref 1
(FA1)		  MUNANA  Stop solution = 0.1 M glycine pH 10.7,     
 25% EtOH rather than published stop solution  
Document Acrobat
  Dr Alexander Klimov    NA-Star  
Document Acrobat

References:

1. Wetherall, N. T., T. Trivedi, J. Zeller, C. Hodges-Savola, J. L. McKimm- Breshkin, M. Zambon, and F. G. Hayden (2003). Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the Neuraminidase Inhibitor Susceptibility Network. J Clin Microbiol. 41:742–750.
2. Gubareva LV, Webster RG, Hayden FG (2002). Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay. Antiviral Res. Jan;53(1):47-61.
3. Blick TJ, Tiong T, Sahasrabudhe A, Varghese JN, Colman PM, Hart GJ, Bethell RC, McKimm-Breschkin JL (1995). Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. Virol. Dec 20;214(2):475-84.