Transcript Interpretation of Raw Data: NA Activity and IC50 Assays

Interpretation of Raw Data:
NA Activity and IC50 Assays
Angie Lackenby
Topics to Address
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Determination of standard virus dose
Validation of NA activity and IC50 assays
Interpretation of NA activity assays
Interpretation of IC50 assays
Determination of Standard Virus Dose
(1)
• Fluorescence unit are RELATIVE
• Raw values are not applicable directly to other instruments
• Standard dose should be within linear enzyme activity
0.10
0.20
0.39
50.00
0.00
0.78
10000.00
1.56
20000.00
3.13
30000.00
6.25
40000.00
12.50
50000.00
100.00
36700
RFU
RFU
60000.00
25.00
70000.00
• Dilute 4-MU (SS) in water to
100mM
• Titrate 4-MU (SS) solution in high
pH stop solution from 100uM to
0.1uM
• Read the fluorescence levels of
the titration
• Plot RFU against 4-MU (SS)
concentration
• Read off the RFU value given by
10uM 4-MU (SS)
4-methylumbilliferone (uM)
• In all virus titrations, the virus
dilution giving this RFU value can
10uM 4-MU (SS)
be used as the standard dose
Determination of Standard Virus
Dose (2)
60000.00
50000.00
40000.00
30000.00
20000.00
10000.00
H3N2
H1N1
Flu B
2048
512
256
1024
Virus Dilution
128
64
32
Dilute 1/6
16
8
4
0.00
2
RFU
10nm
4-MU
10nmol/ml 4-methyumbelliferone
Validation of NA activity and IC50 assays
• Level of fluorescence of the virus control (no drug) in IC50
assay should match standard dose (e.g.36000 RFU)
• Shape of curve/curve fit
• Replicate correlation – set limiting criteria e.g. >30%
difference in IC50?
• Reference strain performance
A
40000
35000
B
IC50: 1.7 vs 3.9
C
30000
25000
IC50: 1.2 vs 5.5
30000
20000
RFU
20000
15000
15000
10000
10000
5000
5000
Oseltamivir Conc (nM)
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
62.50
250
1000
VC
0.0153
0.0610
0.2441
0.9766
3.9063
62.50
250
1000
15.6250
Oseltamivir Conc (nM)
4000
0
0
4000
RFU
25000
Validation: Reference Performance
B
R152
B/Memphis/20/1996
152K
2-4
0.16 – 0.99
0.37 – 4.04
2 (15-20000)
179 – 4000*
2.2 – 97.4
4-6
0.1 – 3.3
0.37 – 4.57
4-6
15.7 - 104
0.27 – 9.23
2-4
0.74 – 3.96
0.29 – 3.36
2 (25-30000)
242 – 4000*
18.77 - 786
45000
40000
RFU
30000
25000
20000
15000
10000
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
62.50
250
1000
5000
0
4000
RFU
35000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
Oseltamivir Conc (nM)
Oseltamivir Conc (nM)
H3N2 Wild Type (119E)
H3N2 Mutant (119V)
H1N1 Wild Type Low Ttire virus
H1N1 Mutant High Resistant
H3N2 Wild Type (292R)
H3N2 Mutant (292K)
Flu B Wild Type
Flu B Mutant (152K)
VC
119V
0.2 – 0.75
0.0153
A/Wuhan/359/1995
138 – 816
0.0610
E119
2 (10-20000)
0.2441
292K
0.1 – 0.67
0.9766
A/Sydney/5/1997
0.14 – 2.46
3.9063
R292
N2
2 (10-20000)
15.6250
Y274
IC50 FL: Z
(+/- 3SD limits)
62.50
A/Texas/36/1991
IC50 FL: O
(+/- 3SD limits)
250
H274
N1
Dilution (RFU
range)
1000
Strain
Designation
Mutation
4000
Subtype
Interpretation of NA Activity
• HA titre should generally correlate with NA activity
– Can indicate resistance even if IC50 is normal
• Recognised subtype specific differences
– In general H1N1< H3N2< Flu B
Both Isolates HA640 and IC50 in normal range
All 3 isolates HA320
60000.00
60000.00
A
50000.00
B
50000.00
40000.00
RFU
30000.00
30000.00
20000.00
20000.00
10000.00
10000.00
Virus Dilution
512
256
128
64
32
16
8
4
512
256
2048
2
Flu B
2048
H3N2
0.00
1024
H1N1
1024
Virus Dilution
128
64
32
16
8
4
0.00
2
RFU
40000.00
Interpretation of IC50 Results
• Recognised subtype specific differences
– Correct sub-tying?
• Recognised drug specific differences
– H3N2 greater sensitivity to oseltamivir than
zanamivir
– Flu B greater sensitivity to zanamivir than
oseltamivir
– H1N1 similar sensitivity to both drugs
• Aside from IC50 value, consider:
– Curve shape (flattened/ non sigmoidal)
– Curve position (shifts to left or right)
– Link HA titre with NA activity and IC50 value
Neuraminidase Inhibitor Concentration (nM)
H3N2
H1N1
Flu B
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
62.50
250
1000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
4000
RFU
IC50 Interpretation: Subtype
specific differences
IC50 Interpretation: Resistant
versus incorrect subtype
A
B
45000
40000
40000
35000
35000
30000
Oseltamivir Conc (nM)
Wrongly Subtyped/Mixed
32nM
Typical H3
0.7nM
Typical Flu B from same
region
25nM
Oseltamivir Conc (nM)
1.9nM
Query
Known H1N1 2.1nM
Known H3N2 0.4nM
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
4000
VC
0.0153
0.0610
0
0.2441
0
0.9766
5000
3.9063
5000
15.6250
10000
62.50
10000
250
15000
1000
15000
62.50
20000
250
20000
25000
1000
RFU
25000
4000
RFU
30000
Interpretation of IC50 data: True
Outliers
40000
35000
30000
20000
15000
10000
5000
Oseltamivir Conc (nM)
Test 1= IC50 1150
Typical H1N1 = IC50 2.14
Test 2 = IC50 825
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
62.50
250
1000
0
4000
RFU
25000