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I have a question with regard to method of calculations for plasma
protein binding assay in drug discovery setting. From the literature
it appears that both area ratio (having established linearity in
response) and calibration curve method have been used to calculate Fu
or % binding.
We did rat and human plasma protein binding assay (equilibrium
dialysis) for 15 compounds from different scaffolds (and different
projects too) along with warfarin and verapamil as assay controls. We
did this assay twice and calculated PPB using area ratio method in
first run (prior to linearity assessment) and calibration curve method
in second run. We didnt observe any significant difference between the
% binding values calculated by both the methods (for example 95.6% vs
95.4). The compound concentration in the assay was 2uM (roughly around
1000ng/ml). Due to limited resources and lack of automation, we are
thinking of using area calculation method instead of running 6-8 point
calibration curves and QCs . The purpose of our running this assay is
to rank order the compounds.
I would like to know the views of group on this.
Thanks
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Having a standard curve gives you the opportunity to check for
recovery, and avoid any non-linear response (in case you are using LC/
MS_MS)
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You could do that if you are sure that the compound response is the
same in the different medium (buffer in the receptor and plasma in the
donor compartment). For the equilibrium dialysis experiment, you need
to quantify your compound in plasma and in buffer. What we use here,
is a dilution of all the samples in order to have the same final
composition : dilution of the plasma samples with blank buffer and
dilution of buffer samples with plasma (one to one dilution).
After that you could do one calibration curve in plasma diluted with
buffer, or use area response. With this protocol, the bias is reduced
as much as possible. It's important if you are doing your assay by LC-
MS/MS.
The other main point could be the linearity of the response,
especially with LC-MS/MS, and you could reduced it by diluting the
samples from the donor compartment.
Fabrice Guillet
XENOBLIS
France
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