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

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] 1400 kDa) enhanced efficiency, but dextran sulfate (average MW 8 kDa) 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 263 K. 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.

► Effects of sulfated polysaccharides on cell-PMCA of vCJD were studied. ► Enhancing effects depended on additives and on their molecular weight. ► The effect of heparin on cell-PMCA was prion strain dependent. ► Detection limit of cell-PMCA of vCJD with heparin was 10−10 dilution of the brain.