TY - JOUR
T1 - Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease
AU - Yokoyama, Takashi
AU - Takeuchi, Atsuko
AU - Yamamoto, Miyuki
AU - Kitamoto, Tetsuyuki
AU - Ironside, James W.
AU - Morita, Masanori
PY - 2011/7/8
Y1 - 2011/7/8
N2 - Highly sensitive in vitro screening tests are required to prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD). Protein misfolding cyclic amplification (PMCA) is a candidate for such a test, but the sensitivity of this method is insufficient. Polyanions were reported to enhance PMCA efficiency, but their effects on vCJD are unclear. We developed a cell-PMCA of vCJD, wherein cell lysate containing exogenously expressed human PrP was used as substrates, to investigate the effects of various sulfated polysaccharides on amplification efficiency. PrPres amounts after cell-PMCA were analyzed by western blotting. Heparin, dermatan sulfate, and dextran sulfate (average molecular weight [MW] 1400kDa) enhanced efficiency, but dextran sulfate (average MW 8kDa) and a heparin pentasaccharide analog had no effect. Pentosan polysulfate inhibited cell-PMCA reaction. The amplification efficiency of cell-PMCA of vCJD increased to >100-fold per round with heparin. The enhancing effects of heparin on cell-PMCA were seed dependent: it was high for vCJD, low for sporadic Creutzfeldt-Jakob disease, and low to negligible for hamster-adapted scrapie-derived 263K. In multi-round PMCA, signals were detected at earlier rounds with heparin than without heparin, and PrPSc in 10-10 diluted vCJD brain was detected by the sixth round. Heparin-assisted cell-PMCA of vCJD represents a significant step toward detecting very minute amounts of PrPSc in the body fluids of asymptomatic vCJD patients.
AB - Highly sensitive in vitro screening tests are required to prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD). Protein misfolding cyclic amplification (PMCA) is a candidate for such a test, but the sensitivity of this method is insufficient. Polyanions were reported to enhance PMCA efficiency, but their effects on vCJD are unclear. We developed a cell-PMCA of vCJD, wherein cell lysate containing exogenously expressed human PrP was used as substrates, to investigate the effects of various sulfated polysaccharides on amplification efficiency. PrPres amounts after cell-PMCA were analyzed by western blotting. Heparin, dermatan sulfate, and dextran sulfate (average molecular weight [MW] 1400kDa) enhanced efficiency, but dextran sulfate (average MW 8kDa) and a heparin pentasaccharide analog had no effect. Pentosan polysulfate inhibited cell-PMCA reaction. The amplification efficiency of cell-PMCA of vCJD increased to >100-fold per round with heparin. The enhancing effects of heparin on cell-PMCA were seed dependent: it was high for vCJD, low for sporadic Creutzfeldt-Jakob disease, and low to negligible for hamster-adapted scrapie-derived 263K. In multi-round PMCA, signals were detected at earlier rounds with heparin than without heparin, and PrPSc in 10-10 diluted vCJD brain was detected by the sixth round. Heparin-assisted cell-PMCA of vCJD represents a significant step toward detecting very minute amounts of PrPSc in the body fluids of asymptomatic vCJD patients.
KW - Cell-PMCA
KW - Heparin
KW - Prion
KW - SCJD
KW - VCJD
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U2 - 10.1016/j.neulet.2011.04.072
DO - 10.1016/j.neulet.2011.04.072
M3 - Article
C2 - 21565253
AN - SCOPUS:79958731755
VL - 498
SP - 119
EP - 123
JO - Neuroscience Letters
JF - Neuroscience Letters
SN - 0304-3940
IS - 2
ER -