O amyloid; in that case, its replacement would not accelerate the RT-QuIC reaction rate.A key goal in TSE diagnostics is detection of prion seeding activity in blood. Here we found that 82 of plasma Title Loaded From File samples from mice clinically affected with Title Loaded From File multiple scrapie strains gave clear positive reactions. However, in the remainder of the mice the plasma seeding activity levels appeared to be near the detection limit. This could be due to naturally low plasma PrPSc concentrations, or to the presence of eQuIC inhibitors in plasma. Nevertheless, negative control samples gave no spontaneous conversion of the substrate 1531364 within 55 h. Further work will be needed to determine if additional gains in sensitivity can be made without increasing the occurrence of false positive reactions. Our comparison of the WT and GPI2 PrP seeds revealed a curious discordance between seed concentration and reaction speed. The reason for the markedly shorter lag phases of RTQuIC reactions seeded with infected brain from GPI2 mice is unclear. Previous work has shown that for a given type of prion seed, lag phases tend to be inversely correlated with seed concentration in RT-QuIC reactions [41,43]. However, end-point dilution QuIC indicated that the seed, or SD50, concentrations in the brains of the GPI2 and WT mice that we examined were indistinguishable for a given strain. End-point dilution RT-QuIC should measure primarily the concentration, rather than the relative seeding capacity, of individual seed particles. Clearly, however, PrPSc seed particles can vary widely in size [13,31] and presumably other characteristics such as seeding activity per particle [13]. For example larger particles, such as plaques or bundles of fibrils, could have many more seeding surfaces than individual fibrils, protofilaments, or small oligomeric seeds. Given that PrPSc in GPI2 PrP transgenic mice accumulates exclusively in the form of large amyloid fibrils and plaques, we suspect that the average seed particle is larger, with more seeding surfaces, than those in WT brain homogenates. This higher per-particle seeding activity could support faster RT-QuIC kinetics for a given overall seed particle concentration. Alternatively, or additionally, the lack of GPI anchors and/or glycans on the GPI2 PrPSc may allow better access of rPrPSen substrate molecules to seeding sites on PrPSc particles, thus improving the rate of conversion per unit seed in the reaction.Figure 9. eQuIC detection of RML PrPSc spiked into mouse plasma 1662274 without substrate replacement. A 561026 dilution of NBH or 561024 to 5610213 dilutions of RML infected brain tissue containing from ,20 pg to 2 ag of PrPRes, respectively, were spiked into 0.2 mL of mouse plasma. PrPSc was immunoprecipitated using 15B3-coated beads and a portion of the beads was used to seed quadruplicate eQuIC reactions. moPrPSen 90?231 was used as a substrate in all reactions. The mean ThT fluorescence of the four replicates is shown. doi:10.1371/journal.pone.0048969.gRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 10. eQuIC detection of endogenous PrPSc in plasma from clinically ill, scrapie-infected mice. (A) plasma samples from five RMLinfected, four 79A-infected and three aliquots of plasma pooled from multiple uninfected mice were analyzed. (B) Plasma sample analyses from 22Linfected WT and GPI2 mice (one each) and pooled plasma from uninfected mice (normal). Endogenous PrPSc was immunoprecipitated using 15B3coated beads and a portion of the beads wer.O amyloid; in that case, its replacement would not accelerate the RT-QuIC reaction rate.A key goal in TSE diagnostics is detection of prion seeding activity in blood. Here we found that 82 of plasma samples from mice clinically affected with multiple scrapie strains gave clear positive reactions. However, in the remainder of the mice the plasma seeding activity levels appeared to be near the detection limit. This could be due to naturally low plasma PrPSc concentrations, or to the presence of eQuIC inhibitors in plasma. Nevertheless, negative control samples gave no spontaneous conversion of the substrate 1531364 within 55 h. Further work will be needed to determine if additional gains in sensitivity can be made without increasing the occurrence of false positive reactions. Our comparison of the WT and GPI2 PrP seeds revealed a curious discordance between seed concentration and reaction speed. The reason for the markedly shorter lag phases of RTQuIC reactions seeded with infected brain from GPI2 mice is unclear. Previous work has shown that for a given type of prion seed, lag phases tend to be inversely correlated with seed concentration in RT-QuIC reactions [41,43]. However, end-point dilution QuIC indicated that the seed, or SD50, concentrations in the brains of the GPI2 and WT mice that we examined were indistinguishable for a given strain. End-point dilution RT-QuIC should measure primarily the concentration, rather than the relative seeding capacity, of individual seed particles. Clearly, however, PrPSc seed particles can vary widely in size [13,31] and presumably other characteristics such as seeding activity per particle [13]. For example larger particles, such as plaques or bundles of fibrils, could have many more seeding surfaces than individual fibrils, protofilaments, or small oligomeric seeds. Given that PrPSc in GPI2 PrP transgenic mice accumulates exclusively in the form of large amyloid fibrils and plaques, we suspect that the average seed particle is larger, with more seeding surfaces, than those in WT brain homogenates. This higher per-particle seeding activity could support faster RT-QuIC kinetics for a given overall seed particle concentration. Alternatively, or additionally, the lack of GPI anchors and/or glycans on the GPI2 PrPSc may allow better access of rPrPSen substrate molecules to seeding sites on PrPSc particles, thus improving the rate of conversion per unit seed in the reaction.Figure 9. eQuIC detection of RML PrPSc spiked into mouse plasma 1662274 without substrate replacement. A 561026 dilution of NBH or 561024 to 5610213 dilutions of RML infected brain tissue containing from ,20 pg to 2 ag of PrPRes, respectively, were spiked into 0.2 mL of mouse plasma. PrPSc was immunoprecipitated using 15B3-coated beads and a portion of the beads was used to seed quadruplicate eQuIC reactions. moPrPSen 90?231 was used as a substrate in all reactions. The mean ThT fluorescence of the four replicates is shown. doi:10.1371/journal.pone.0048969.gRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 10. eQuIC detection of endogenous PrPSc in plasma from clinically ill, scrapie-infected mice. (A) plasma samples from five RMLinfected, four 79A-infected and three aliquots of plasma pooled from multiple uninfected mice were analyzed. (B) Plasma sample analyses from 22Linfected WT and GPI2 mice (one each) and pooled plasma from uninfected mice (normal). Endogenous PrPSc was immunoprecipitated using 15B3coated beads and a portion of the beads wer.
