Ally diluted 2-fold and 4-fold. A final concentration of 20 mg/mL PK was used to digest brain homogenates. Bands were detected with monoclonal antibody 6D11 as described in materials and methods. doi:10.1371/journal.pone.RG 7422 0048969.gRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 5. Total protein staining of seeded conversion products from GPI2 and WT mice inoculated with RML or normal (NBH) BH. 561026 dilutions were used to seed RT-QuIC reactions containing moPrPC23?31 substrate. Reaction products were PK digested (+) at final concentration of 10 mg/mL, or not (2) and analyzed by SDS-PAGE. The gel was stained with a total protein stain (Deep Purple). Lanes 1,3: no PK and PK-treated WT uninfected products; Lanes 2,4: no PK and PKtreated WT RML infected products. Lane 5,7: no PK and PK-treated GPI2 uninfected products. Lane 6,8: no PK and PK-treated GPI2 RML infected products. The oval indicates the weak ,18 kDa bands while the bracket represents the 12, 13 and 14 kDa bands in the PK-digested products of the scrapie-seeded reactions (lanes 4 and 8). doi:10.1371/journal.pone.0048969.g005 Figure 4. Seeding activity and Log SD50 in GPI2 and WT mice infected with multiple scrapie strains. RT-QuIC reactions were seeded with 561027 and 561028 brain dilution from WT and GPI2 mice infected with 22L (A) and ME7 (B) strains; 561028 and 561029 brain dilutions from WT and GPI2 mice infected with RML were compared in (C). moPrPSen 23?31 was used as substrate in all reactions. doi:10.1371/journal.pone.0048969.gRT-QuIC reactions, but, curiously, did not show this tendency 1527786 in eQuIC reactions. We speculate that the presence of antibody coated beads and/or the altered kinetics of the eQuIC mightpositive replicates, two with 3/4 and two with 2/4) while the two remaining scrapie-affected mice gave 1/4 positive replicates (Figure 10A). In contrast, tests of 4 negative control mice gave 0/4 positive replicates, while 1 negative control specimen gave 1/4 positives, with the latter being an apparent false positive occurring late in the reaction (over 55 h). We also got similarly positive reactions (all 4/4 positive replicates) from plasma samples from clinically affected WT and GPI2 mice inoculated with 22L scrapie (Figure 10B). Collectively, our data showed the ability of the 15B3based eQuIC to detect a variety of different mouse-adapted scrapie strains endogenous to plasma in the clinical phase of disease.DiscussionHere we demonstrate the in vitro amplified detection of mouseadapted scrapie strains by RT-QuIC and e-QuIC assay. In general, the use of full-length moPrPSen23?31 and low NaCl concentrations GDC-0068 chemical information allowed rapid and sensitive mouse seed amplification with a very low incidence of false positive reactions in the RTQuIC. The truncated moPrPSen90?31 substrate tended to undergo spontaneous (prion seed-independent) conversion inFigure 6. PrPRes levels in synaptosomal 24786787 fractions from 263Kand 139A-infected 101LL mice by immunoblotting. Lane 1: no PK 101L 263K sample. Lanes 2?: PK-treated 101L 263K samples undiluted and serially diluted 3-fold and 9-fold. Lane 5: no PK 101L 139A sample. Lanes 6?1: PK-treated 101L139A samples undiluted and serially diluted 3-fold, 9-fold, 27-fold, 81-fold and 243-fold. A final concentration of 100 mg/mL PK was used to digest synaptosomal fractions as described in Materials and Methods. Samples were serially diluted in sample buffer. Bands were detected with monoclonal antibody 6D11. doi:10.1371/journal.pone.0048969.gRT-QuIC and eQuI.Ally diluted 2-fold and 4-fold. A final concentration of 20 mg/mL PK was used to digest brain homogenates. Bands were detected with monoclonal antibody 6D11 as described in materials and methods. doi:10.1371/journal.pone.0048969.gRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 5. Total protein staining of seeded conversion products from GPI2 and WT mice inoculated with RML or normal (NBH) BH. 561026 dilutions were used to seed RT-QuIC reactions containing moPrPC23?31 substrate. Reaction products were PK digested (+) at final concentration of 10 mg/mL, or not (2) and analyzed by SDS-PAGE. The gel was stained with a total protein stain (Deep Purple). Lanes 1,3: no PK and PK-treated WT uninfected products; Lanes 2,4: no PK and PKtreated WT RML infected products. Lane 5,7: no PK and PK-treated GPI2 uninfected products. Lane 6,8: no PK and PK-treated GPI2 RML infected products. The oval indicates the weak ,18 kDa bands while the bracket represents the 12, 13 and 14 kDa bands in the PK-digested products of the scrapie-seeded reactions (lanes 4 and 8). doi:10.1371/journal.pone.0048969.g005 Figure 4. Seeding activity and Log SD50 in GPI2 and WT mice infected with multiple scrapie strains. RT-QuIC reactions were seeded with 561027 and 561028 brain dilution from WT and GPI2 mice infected with 22L (A) and ME7 (B) strains; 561028 and 561029 brain dilutions from WT and GPI2 mice infected with RML were compared in (C). moPrPSen 23?31 was used as substrate in all reactions. doi:10.1371/journal.pone.0048969.gRT-QuIC reactions, but, curiously, did not show this tendency 1527786 in eQuIC reactions. We speculate that the presence of antibody coated beads and/or the altered kinetics of the eQuIC mightpositive replicates, two with 3/4 and two with 2/4) while the two remaining scrapie-affected mice gave 1/4 positive replicates (Figure 10A). In contrast, tests of 4 negative control mice gave 0/4 positive replicates, while 1 negative control specimen gave 1/4 positives, with the latter being an apparent false positive occurring late in the reaction (over 55 h). We also got similarly positive reactions (all 4/4 positive replicates) from plasma samples from clinically affected WT and GPI2 mice inoculated with 22L scrapie (Figure 10B). Collectively, our data showed the ability of the 15B3based eQuIC to detect a variety of different mouse-adapted scrapie strains endogenous to plasma in the clinical phase of disease.DiscussionHere we demonstrate the in vitro amplified detection of mouseadapted scrapie strains by RT-QuIC and e-QuIC assay. In general, the use of full-length moPrPSen23?31 and low NaCl concentrations allowed rapid and sensitive mouse seed amplification with a very low incidence of false positive reactions in the RTQuIC. The truncated moPrPSen90?31 substrate tended to undergo spontaneous (prion seed-independent) conversion inFigure 6. PrPRes levels in synaptosomal 24786787 fractions from 263Kand 139A-infected 101LL mice by immunoblotting. Lane 1: no PK 101L 263K sample. Lanes 2?: PK-treated 101L 263K samples undiluted and serially diluted 3-fold and 9-fold. Lane 5: no PK 101L 139A sample. Lanes 6?1: PK-treated 101L139A samples undiluted and serially diluted 3-fold, 9-fold, 27-fold, 81-fold and 243-fold. A final concentration of 100 mg/mL PK was used to digest synaptosomal fractions as described in Materials and Methods. Samples were serially diluted in sample buffer. Bands were detected with monoclonal antibody 6D11. doi:10.1371/journal.pone.0048969.gRT-QuIC and eQuI.
