Journal: Viruses

Article Title: Kinetics of Abnormal Prion Protein in Blood of Transgenic Mice Experimentally Infected by Multiple Routes with the Agent of Variant Creutzfeldt–Jakob Disease

doi: 10.3390/v15071466

Figure Lengend Snippet: Direct comparison of assay sensitivity for HerdChek (HC) PrP TSE test and Western blot. PMCA products were serially diluted 2-fold in 10% normal vole brain. We used 25 µL of each sample for HC PrP TSE tests. After proteinase K digestion, the equivalent of 10 µL of each sample was assayed by Western blot. The top panel also shows colorimetric results for each sample and for internal controls (negative and positive) included in the HC kit. The horizontal dotted line corresponds to the assay threshold (O.D. 0.31) . The Western blot lanes were aligned with corresponding HC samples. NVB, normal vole brain (PMCA substrate). Representative results from a total of three independent experiments.

Article Snippet: We measured levels of PMCA products using a colorimetric enzyme-linked immunosorbent assay (ELISA-like) called IDEXX HerdChek BSE-Scrapie Antigen Test kit (HC PrP TSE test) that detects PrP TSE as previously described [ ].

Techniques: Comparison, Western Blot

Journal: Prion

Article Title: A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

doi: 10.1080/19336896.2016.1166324

Figure Lengend Snippet: PrPSc analysis of donor animals' brain homogenates. (A) TSE ELISA analysis of caprine scrapie-infected goat and Tg338 mouse brain homogenates for PrPSc. Approximately 300 μg of 10% w/v brain homogenates of caprine scrapie-infected goat (animal IDs: g3558, g30-75, g3950, g4425, and g4428) and Tg338 mouse (animal IDs: m316, m298, and m178) brain homogenates (in duplicate) was assessed for relative levels of PrPSc using a TSE ELISA kit (IDEXX). Scrapie-uninfected goat (animal ID: g4111) and Tg338 mouse (animal ID: m1628) brain homogenates were also included in the ELISA. Average TSE ELISA absorbance values are shown in the y-axis and animal IDs are shown in the x-axis. Cut-off value for the ELISA (—) was determined as described by the manufacturer. Western blot analysis of caprine scrapie-infected goat (B) and Tg338 mouse (C) brain homogenates for PrPres. Caprine scrapie-infected and scrapie-uninfected goat and Tg338 mouse brain homogenates (˜75 μg wet tissue weight for each sample) were incubated with proteinase K (100 µg ml−1 at 37°C for 60 min) and PrPres was detected using PrP mAbs F99/97.6.1 (3.5 µg ml−1). The positions of the molecular mass markers (in kDa) are shown on the left.

Article Snippet: Detection of PrP Sc by ELISA and PrP res by Immunoblotting Accumulations of PrP Sc in inoculated cpRK13 cell lysates and caprine scrapie brain homogenates were evaluated using a commercially available TSE ELISA kit (CWD Antigen test kit EIA, IDEXX Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Incubation

Journal: Prion

Article Title: A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

doi: 10.1080/19336896.2016.1166324

Figure Lengend Snippet: Detection of de novo PrPSc propagation in cpRK13 cells. (A) TSE ELISA analysis of cpRK13 cells inoculated with caprine scrapie-infected goat brain homogenates. Caprine PrPC expressing cpRK13 (black bars) and plasmid control RK13 cells (pcRK13, open bars) were inoculated with scrapie-infected (animal IDs: g3558, g30-75, g3950, g4425, and g4428,) or scrapie-uninfected (animal ID: g4111) goat brain homogenates and cell lysates were prepared 5 weeks post-inoculation. One hundred μl of cpRK13 or pcRK13 cell lysate was loaded into each well (in triplicate) and relative levels of PrPSc accumulations were evaluated using a TSE ELISA kit (IDEXX). Average TSE ELISA absorbance values with corresponding standard deviations are shown in the y-axis and animal IDs are shown in the x-axis. Cut-off value for the ELISA (—) was determined as described by the manufacturer. (*, P<0.01). (B) Detection of PrPres in cpRK13 cells inoculated with caprine scrapie-infected brain isolates. Western blot assays were performed with cpRK13 cell lysates prepared from scrapie-infected (animal IDs: g3558, g30-75, g3950, g4425, and g4428) or scrapie-uninfected goat brain homogenates (animal ID: g4111). Twenty µl of inoculated cpRK13 or pcRK13 cell lysates were incubated with proteinase K (100 µg ml−1 at 37°C for 60 min) and PrPres was detected using a mixture of PrP mAbs F99/97.6.1. (3.5 µg ml−1) and P4 (0.2 µg ml−1). The positions of the molecular mass markers (in kDa) are shown on the left. (C) and (D). In situ detection of PrPSc accumulation in cpRK13 cells by IHC. Caprine scrapie prion propagation in cpRK13 cells was also assessed by IHC with HistoGel method. PrPSc immunolabeling was clearly visible in cpRK13 cell (D) but not in pcRK13 cells (C) following inoculation with scrapie goat brain homogenates (animal ID: g4428). PrPSc (dark red) in the cells were identified using PrP mAb SAF84.

Article Snippet: Detection of PrP Sc by ELISA and PrP res by Immunoblotting Accumulations of PrP Sc in inoculated cpRK13 cell lysates and caprine scrapie brain homogenates were evaluated using a commercially available TSE ELISA kit (CWD Antigen test kit EIA, IDEXX Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Plasmid Preparation, Control, Western Blot, Incubation, In Situ, Immunolabeling

Journal: Prion

Article Title: A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

doi: 10.1080/19336896.2016.1166324

Figure Lengend Snippet: Permissibility of cpRK13 cells to brain-derived, Tg338 mouse-passaged heterozygous PRNP classical caprine scrapie prion isolates. Caprine PrPC expressing cpRK13, and plasmid control (pcRK13) cells were inoculated with brain homogenates prepared from scrapie-infected second passaged Tg338 mice inoculated with a caprine PRNP haplotype 1,1 (animal ID: g3538, m298), haplotype 2,3 (animal IDs: g30-75, m316), haplotype 1,4 (animal ID: g3558, m178) or uninoculated mouse (animal ID: m1628, neg ctl). One hundred μl of cpRK13 (black bars), or pcRK13 (open bars) cell lysate was loaded into each well (in triplicate) and relative levels of PrPSc accumulations were evaluated using a TSE ELISA kit (IDEXX). Average TSE ELISA absorbance values with corresponding standard deviations are shown in the y-axis and animal IDs are shown in the x-axis. Cut-off value for the ELISA (—) was determined as described by the manufacturer (*, P<0.01).

Article Snippet: Detection of PrP Sc by ELISA and PrP res by Immunoblotting Accumulations of PrP Sc in inoculated cpRK13 cell lysates and caprine scrapie brain homogenates were evaluated using a commercially available TSE ELISA kit (CWD Antigen test kit EIA, IDEXX Laboratories).

Techniques: Derivative Assay, Expressing, Plasmid Preparation, Control, Infection, Enzyme-linked Immunosorbent Assay