1. Interpretation of Raw Data: NA Activity and IC50 Assays Angie Lackenby

2. Topics to Address Determination of standard virus dose Validation of NA activity and IC50 assays Interpretation of NA activity assays Interpretation of IC50 assays

3. 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 36700 RFU 10uM 4-MU (SS) 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) In all virus titrations, the virus dilution giving this RFU value can be used as the standard dose

4. Determination of Standard Virus Dose (2) 10nm 4-MU Dilute 1/6

5. 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 IC50: 1.7 vs 3.9 IC50: 1.2 vs 5.5 A B C

6. Validation: Reference Performance SubtypeMutation Strain Designation Dilution (RFU range) IC50 FL: O (+/- 3SD limits) IC50 FL: Z (+/- 3SD limits) N1 H274 A/Texas/36/1991 2 (10-20000)0.14 – 2.460.1 – 0.67 Y2742 (10-20000)138 – 8160.2 – 0.75 N2 R292 A/Sydney/5/1997 2-40.16 – 0.990.37 – 4.04 292K2 (15-20000)179 – 4000*2.2 – 97.4 E119 A/Wuhan/359/1995 4-60.1 – 3.30.37 – 4.57 119V4-615.7 - 1040.27 – 9.23 BR152 B/Memphis/20/1996 2-40.74 – 3.960.29 – 3.36 152K2 (25-30000)242 – 4000*18.77 - 786

7. 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 AB

8. 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

9. IC50 Interpretation: Subtype specific differences

10. IC50 Interpretation: Resistant versus incorrect subtype 32nM 0.7nM 25nM 1.9nM 2.1nM 0.4nM A B

11. Interpretation of IC50 data: True Outliers