Efficient propagation of variant Creutzfeldt-Jakob disease prion protein using the cell-protein misfolding cyclic amplification technique with samples containing plasma and heparin.
BACKGROUND
To prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD) between humans via blood products or transfusion, highly sensitive in vitro screening tests are necessary. Protein misfolding cyclic amplification (PMCA) is one such candidate test. However, plasma has been reported to inhibit the PMCA reaction. Therefore, we investigated the cell-PMCA conditions that permit vCJD prion amplification in the presence of plasma.


METHODS
Cell-PMCA of vCJD samples was performed by adding various final concentrations of pooled plasma, citrate-phosphate-dextrose (CPD), albumin, globulin, or pooled plasma treated with ion exchangers. After heparin and plasma concentrations were optimized, multiround cell-PMCA was performed.


RESULTS
When 1% to 50% of pooled plasma was added to heparinized cell-PMCA, amplification efficiency showed a double-peaked profile at less than 1% and 40% final plasma concentrations, indicating that plasma contains not only PMCA inhibitors but also promoters. Intravenous globulin did not inhibit cell-PMCA, but the protein G-bound fraction did. CPD, albumin-depleted plasma, and the unbound fraction of anion-exchange chromatography inhibited cell-PMCA, but albumin and the unbound fraction of the cation-exchange chromatography did not. The detection limit of abnormal prion protein in multiround cell-PMCA, when maintaining the final plasma concentration at 40% at each round, was 10(-10) dilutions of a vCJD brain specimen.


CONCLUSIONS
We have established a novel cell-PMCA format in the presence of plasma without any pretreatment, where vCJD prion protein was amplified at comparable levels to that found without plasma. Our data suggest the feasibility of cell-PMCA as a practical blood test for vCJD prions.
Research and Development Division, Japan Blood Products Organization, Tokyo, Japan.
Masatoshi
M
Oshita
Research and Development Division, Japan Blood Products Organization, Tokyo, Japan.
Takashi
T
Yokoyama
Research and Development Division, Japan Blood Products Organization, Tokyo, Japan.
Yumiko
Y
Takei
Department of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Miyagi Prefecture, Japan.
Atsuko
A
Takeuchi
National CJD Research and Surveillance Unit, Centre for Clinical Brain Sciences, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
James W
JW
Ironside
Department of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Miyagi Prefecture, Japan.
Tetsuyuki
T
Kitamoto
Research and Development Division, Japan Blood Products Organization, Tokyo, Japan.
Masanori
M
Morita
D015415:Biomarkers / Q000378:metabolism*; D058731:Blood Safety / Q000379:methods*; D007562:Creutzfeldt-Jakob Syndrome / Q000097:blood / Q000175:diagnosis*; D006403:Hematologic Tests; D006493:Heparin; D006801:Humans; D010949:Plasma; D011328:Prions / Q000097:blood*
D015415:Biomarkers; D011328:Prions; D006493:Heparin