Dear Chris,
Mary Jo Schmerr (USDA/ARS, Ames, Iowa) and I have developed a test for
abnormal prions (PrPsc, etc.) that can easily detect 2 pg/ml of the protein
in blood and other fluids. This compares with about 100 pg/ml for the
current generation of Western blot-based assays. One method you might try
would be to run your digestion with methodical changes to different variables
(time, temperature, etc.) and work up aliquots with our assay. It's a
competition immunoassay, and so will respond until the Proteinase K has
chewed up the region corresponding to the epitope used to raise the antibody
involved.
Proteinase K is used for digestion of PrPc because it digests proteins all
the way down to dipeptides and the like. That insures the destruction of the
epitope region of the PrPc protein, leaving only the PrPsc protein possessing
a suitable antigenic region.
To implement our test, you'll need the following:
1) Consumables: a) A SPE cartridge from our company ($ 5.00 each); b) A
fluorescein-tagged peptide corresponding to an epitope of the prion protein;
c) A polyclonal antibody against the peptide (which also crossreacts with all
prions tried to date).
2) Equipment: Either a CE apparatus or an HPLC system with a
fluorescence detector (preferably Laser Induced Fluorescence). If you use
the HPLC system, then you'll need one of our HPLC columns too ($ 475).
Sample throughput is about 12' between injections for CE and 3-4' for HPLC.
There was a symposium on this subject at Pittcon 2001. Stan Prusiner gave a
lecture there. One lecture concerned modifications to the Proteinase K
digestion that made PrPsc resistant to Proteinase K attack but accelerated
the digestion of PrPc.
We can help you implement this assay. Give us a call if you're interested.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: (410) 992-5400 (NOTE: On 4/9 and 4/10/2001, call me at (609)
921-9527)
*************************************
> Subj:protease test
> Date:4/8/2001 10:30:49 PM Eastern Daylight Time
> From: turck@itsa.ucsf.edu (Christoph W. Turck)
> Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
> Facilities)
> To: abrf@aecom.yu.edu (Recipients of ABRF List)
>
>
> I am interested in establishing a test for protease-resistance of
> proteins. No, I am not planning to get involved in BSE research, but I know
> that there is an assay for testing the protease-resistant abnormal isoform
> of the prion protein PrPC that relies on a digest with proteinase K.
>
> Can anybody suggest protocols for performing such an assay? Also, is
> proteinase K the best enzyme for such an assay or does it just happen to
> work best for the prion protein PrPC?
>
> Thanks,
>
> Chris (turck@itsa.ucsf.edu)
>
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