2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 39
endpoint is recorded as well 9.5. The unitage would then be the geometric mean Data were fit to a sigmoidal dose response curve (variable slope) using the
of the values obtained at wells 9 and 10. In the above example, well 8 would be GraphPad Prism software package. The EC
50
values were determined for each
80,000 U/ml, well 9 would be 160,000 U/ml and well 9.5 would be 113,137 sample as the dilution from the stock required to give 50% protection. From the
U/ml. Generally, within a single assay, the endpoint will be within one well for known unitage of the Lab Standard, it is possible to determine the IFN
replicates. The calculated values for each replicate are then arithmetically concentration required to protect 50% of the cells. In this case, 1.1 U/ml gives
averaged to obtain the U/ml for the sample. Thus, a sample that gave endpoints 50% protection. Thus at a 8.2 x 10
6
dilution Sample 1 should have 1.1 U/ml since
of 8 and 9 would have unitage of 120,000 (Average of 80,000 and 160,000). that is the concentration that gives 50% protection. The U/ml is then the Std
If the concentration of the IFN in the samples is known, it is also then U/ml EC
50
times this dilution. Again, knowing the concentration of the sample,
possible to calculate the specific activity. To do this, one divides the U/ml by the it is possible to determine the pg/ml which gives 50% protection and the
concentration in mg/ml, the results are then expressed as U/mg. In the above Specific Activity of the samples.
example, if the concentration of the sample is 0.0004 mg/ml the specific Since bioassays are inherently complex assays which vary due to the
activity would be 3.0 x 10
8
U/mg (calculated as 120,000 U/ml divided by metabolic state of the cells, the ability of the IFN to protect the cells, and the
0.0004 mg/ml), a value which is typical for an IFN-α or IFN-β sample. virus replication, there will be greater variability in these assays than mass-
A further analysis can be done by solublizing the dye in the cells using a based assays such as ELISAs. For example, during the calibration of the
solution, such as 70% MeOH. This allows determination of the absorbance in the international standard for IFN-β interferon which was a multi-site, multi-assay
wells using a plate reader. Again, using the cell control as 100% protection and study, the geometric CVs obtained were on the order of 60-80%. Generally in
the virus control as 0% protection a curve can be plotted and the EC
50
these assays, results between experimental runs and operators can vary by
determined either in U/ml or pg/ml. about 1/2 log from the actual value. Thus, in order to obtain solid numbers,
multiple runs are required. With experience and careful control, the CV values
GraphPad Prism Analysis of IFN CPE Assay
can be reduced.
Below are the results from 3 different operators over a 4 week period for the
Lab Standard in the A549/EMCV assay. The nominal titer (determined by 20
runs versus the international standard) is 4276 U/ml. In this study the average
potency is 3996 and the CV is 33%. The geometric mean is 3885 and the
i
o
n
geometric CV is 13%.
e
ct
In summary, there are multiple methods available to measure the potency of
a particular IFN preparation. The standard assay measures protection from viral
P
r
ot
%
challenge. However there can be significant variation in the results obtained in
different assay systems with a particular interferon.
[IFN] log (dilution)
Sample 1 Sample 2 Std Std U/ml
EC
50
Dilution 8.2 x 10
6
1.1 x 10
8
44712 1.1
U/ml 8.9 x 10
6
1.2 x 10
8
48736 ---
[Stock] pg/ml 5.0 x 10
7
2.5 x 10
8
1.4 x 10
5
---
EC
50
pg/ml 6.1 2.4 3.5 ---
SA U/mg 1.8 x 10
8
4.6 x 10
8
3.5 x 10
8
---
Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7
U/ml 2.80 x 10
3
6.31 x 10
3
4.53 x 10
3
3.97 x 10
3
3.31 x 10
3
2.90 x 10
3
4.14 x 10
3
www.interferonsource.com Tel: +1 732-777-9123
I
Fax: +1 732-777-9141
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