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Ic50 Calculation and Analysis
Ic50 Calculation and Analysis
NA: Neuraminidase
NI: Neuraminidase Inhibitor
RFU: Relative Fluorescence Units
RLU: Relative Luminescence Units
VC: Virus Control
Determining NI IC50 Values
Upper Asymptote
UK Surveillance
40000
A/Eng/683/2007 #1
A/Eng/683/2007 #2
A/Eng/684/2007 #1
Response
A/Eng/684/2007 #2
20000 A/Eng/685/2007 #1
A/Eng/685/2007 #2
A/Eng/686/2007 #1
A/Eng/686/2007 #2
0
0.01 0.1 1 10 100 1000
Oseltamivir
IC50 Calculation: Point to Point
IC50 Calculation: Comparison of IC50
values from different calculation methods
Determining IC50 Values: Sources of
Variation
• Method of calculation
• Using point-to-point or curve fitting software
• Choice of curve fitting software used
• Intra-assay variation
• Difference between 2 or more replicates
• Inter-assay variation
Difference in calculated value for a given isolate in multiple assays
IC50 Calculation: Comparison of Point
to Point versus curve-fitting
Equivalent values
in SMAD analysis
Using Box and Whisker Analysis:
Example Data
Graphpad Prism Output Excel Output
Median 1.08
Mild outlier lower fence (1.5*IQR) 0.55
Extreme outlier lower fence (3*IQR) 0.34
Mild outlier higher fence (1.5*IQR) 1.95
Extreme outlier higher fence (3*IQR) 3.14
Do we need to log-transform?
Log10 Data
Median 0.72
Robust SD 0.50
Minor Outlier (1.65SD) 1.55
Outlier (3SD) 2.23
Impact of Excess Numbers of Outliers
• If the data have a large number of outliers, both SMAD and B+W
struggle to determine sensible cut offs.
• As resistant virus is very clearly different, these values can be
removed prior to analysis to allow sensible calculations of cut offs
for the remaining data.
• Below, data is shown for H1N1 in 2007/8, when sensitive and
resistant virus co-circulated.
Cut offs calculated do not accurately apply to the sensitive IC50 data
10.00
3SD
1.65 SD
IC50
1.00
Median
0.10
2006/07
2005/06
100.00
10.00
3SD
IC50
1.65 SD
1.00
Median
0.10
2006/07
2005/06
Trends in IC50 Data: Box and
Whisker
H1N1 isolates 2004-2007 EU+ UK: Oseltamivir
5.0
4.5
4.0
IC50 (nM)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
H1N1 EU+UK 04-05 H1N1 EU+UK 05-06 H1N1 EU+UK 06-07
Summary