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The following message was posted to: PharmPK
Hi All,
I would like to ask for some help on a PK study we just did in mice. We
gave 25 micrograms of a recombinant protein to mice intravenously and
took blood samples via cardiac puncture starting at 30' post dose. By
our ELISA assay, the plasma levels for 30 and 60 minutes were in excess
of 35ug/ml--that is more protein that we injected if one multiplies the
concentration by the presumed blood volume of a mouse. We have done
what we think are proper controls for the ELISA (spiking protein into
100% mouse serum and making dilutions in serum, buffer and 10% serum)
and the standard curves are all identical.
Thanks in advance
Mark Perrone
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The following message was posted to: PharmPK
Assuming the quanitation of your recombinant protein was correct (i.e.,
you really injected 25ug/mouse), then based on the below assumptions
there could be many reasons why your ELISA is detecting more protein:
[Assuming the average mass of a mouse is 20g (0.020kg), and assuming
the total blood volume of a mouse is 10% of it's mass, the total blood
volume of a 20g mouse is 2.0mLs*. The calculated blood Cmax should be
25ug/2.0mLs = 12.5ug/mL and your captured plasma Cmax would be
concentrated ~2.5-fold (due to the fact that in order to obtain plasma
you have to spin out the red blood cells which reduces the total volume
and increase the concentration of the protein somewhere between 50% to
66% (average = 58%). The calculated Plasma Cmax in your ELISA should be
31.25ug/mL (25ug/0.8mL plasma).]
(1). Did you include control wells in your ELISA that included all mAbs
in the procedure, but exclude your recombinant protein (i.e., is the
secondary mAb you are using in your ELISA binding to your primary mAb
non-specifically, etc...)?
(2). Did your ELISA development for this experiment include determining
the background signal effects of normal naive mouse plasma on your
ELISA (possibly there is something in your mouse plasma that is
interfering with this ELISA)?
(3). Was your recombinant protein quanitated correctly or does your
recombinant protein contain contaminants that could be interfering with
the ELISA (i.e., what does size exclusion chromatography, native Gels,
SDS-PAGE, and/or westerns reveal about the purity of your protein,
etc...)? Is your recombinant protein contaminated with LPS (can be very
sticky and interfere with ELISA's in general).
Also, I am interested to know the reasons why you chose to inject a
straight 25ug/mouse rather than an amount based on body mass (mg/kg)?
*only 1/2 of the total blood volume will be reasonably expected to be
withdrawn - ~1.0mL blood max from a 20g mouse.
Jennifer D. Thompson
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The following message was posted to: PharmPK
Hi Dear All,
I have similar problem within the first several minutes after
administration of the recombinant protein should the blood concentration
of the recombinant protein be higher than theoretically diluted by the
same amount of body blood? My results showed the first 10 minutes serum
concentrations are higher than it is if diluted with the same amount of
total body serum. Is it because the effective circulation blood volume
is lower than the theoretical total body blood. Please help me.
Thank you very much
with the best regards
Weidong Li
--
The following message was posted to: PharmPK
This is a very interesting topic for me, as I am doing almost the same
thing except using canines rather than mice. I am a student and I have
some questions.
First how do you separate the serum( keep it for how long at room
temperature and centrifugation condition)? Do you think the protein can
be distributed quickly enough in the whole blood volumn(under anesthesia
condition the blood pressure is lower and the circulation of the mouse
is abnormal)? Do you use intravenous bolus injection? How about the
volumn of the recombinant protein?
If too much volumn of the protein for the mouse its heart can't
effectivly pump and cause the protein distributed not effectively.
I also have some questions for my own experiment, please help me. How
to prove the recombinant protein can be metabolised( or catabolized) by
the liver or kidney? How to prove the molecule can or can't pass
through the CSF blood barrier and blood brain barrier? Do you think
just by using the pharmacokinetics profile of that (maybe not be so
useful) recombinant protein I can publish a paper?
If I can publish it what kind of journal I should submit it to ?
Thank you in advance
With the highest regards
Weidong Li
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