Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease

Takashi Yokoyama, Atsuko Takeuchi, Miyuki Yamamoto, Tetsuyuki Kitamoto, James W Ironside, Masanori Morita

Research output: Contribution to journalArticlepeer-review

Abstract

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. PrP(res) 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 PrP(Sc) 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 PrP(Sc) in the body fluids of asymptomatic vCJD patients.
Original languageEnglish
Pages (from-to)119-23
Number of pages5
JournalNeuroscience Letters
Volume498
Issue number2
DOIs
Publication statusPublished - 2011

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