Journal: International Journal of Molecular Sciences

Article Title: Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and miRNAs as Biomarkers for Prion Diseases

doi: 10.3390/ijms22136822

Figure Lengend Snippet: Detection of the pathologic prion protein (PrP Sc /PrP res ) by Dot blot ( A ) and Western blot ( B ) in PMCA reactions seeded with serially diluted (1/5, 1/10 and 1/50) CSF-derived sEVs obtained from scrapie-affected sheep. Immunodetection was performed using the monoclonal Sha31 antibody. ( A ) Representative images of PrP res detection by Dot blot after four PMCA rounds of CSF-derived sEVs from one sheep at clinical stage of scrapie, one sheep at terminal stage, one sheep at preclinical stage and one negative sheep. A brain homogenate from scrapie sheep (10 −3 to 10 −9 diluted) is also shown for comparison. Some dots were subjected to Western blot for PrP res profile detection. ( B ) Representative images of PrP res detection by Western blot after four PMCA rounds of CSF-derived sEVs from one sheep at clinical stage of scrapie (1/10 dilution), one sheep at terminal stage (1/10 dilution), one sheep at preclinical stage (1/50 dilution), one negative sheep (1/10 dilution) and a brain homogenate from scrapie sheep (10 −6 and 10 −7 dilution). A proteinase K-digested classical scrapie isolate (Dawson strain) was used as positive control.

Article Snippet: PrP Sc detection was performed using Sha31 mAb conjugated to horseradish peroxidase (0.06 μg/mL), and ECL substrate (Pierce) was used to reveal peroxidase activity.

Techniques: Dot Blot, Western Blot, Derivative Assay, Immunodetection, Comparison, Positive Control

Journal: PLoS ONE

Article Title: Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

doi: 10.1371/journal.pone.0015839

Figure Lengend Snippet: Mice inoculated with with H-BSE (lanes 1–3 and 5–7 from isolates 01-2604 and 03-2095 respectively), at first (lanes 1, 5) or second passage (lanes 2–3 and 6–7), were compared to mice infected with classical BSE (lanes 4, 8) in panels A–C. At second passage, PrP res exhibited either H-type features, from mice that died later (491 and 504 d.p.i. in lanes 2 and 6 respectively), or “C-BSE like” features (464 and 322 d.p.i. in lanes 3 and 7 respectively). The brain tissue equivalents loaded per lane are indicated (in tenth of mg). D - Glycoform proportions (means +/− standard deviations) of PrP res from H-BSE (isolate 03-2095)-infected mice detected using Sha31 antibody. Mice exhibited PrP res with a molecular mass either similar to that of H-BSE (full squares) or to classical BSE (full diamonds). E - PrP d extracted in the absence of protease digestion and deglycosylated by PNGase treatment from H-BSE infected mice at first (lane 1) or second passage (lanes 2-3 from mice that died at 497 d.p.i. and 464 d.p.i. respectively), compared to a classical BSE control in lane 4 (0.3 mg brain tissue equivalent per lane). Full-length PrP d (FL) and C1 and C2 fragments are indicated. Panels A and B were revealed by monoclonal antibodies Sha31 and 12B2 respectively and panels C and E by HRP-labelled SAF84 antibody. Bars to the left indicate the 29.0, 20.1 and 14.3 kDa marker positions.

Article Snippet: PrP res or PrP d were detected using the monoclonal antibodies anti-PrP Sha31 (1/10 from kit TeSeE sheep/goat Biorad), SAF84 (500 ng/ml)(SPI-Bio, France) or 12B2 (340 ng/ml) against the 144-WEDRYYRE-151, 163-RPVDQY-168 and 88-WGQGG-92 murine PrP sequences respectively.

Techniques: Infection, Control, Bioprocessing, Marker

Journal: PLoS ONE

Article Title: Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

doi: 10.1371/journal.pone.0015839

Figure Lengend Snippet: A - Detection of PrP res from spleens of mice inoculated with H-BSE isolates 01-2604 (lanes 1–3) or 03-2095 (lanes 4–9) at first passage. B - Detection of PrP res from spleens of mice inoculated with H-BSE at third passage in mice inoculated with a brain homogenate with “C-BSE like” PrP res (lanes 4–8). PrP res from the brain of one of these mice (lanes 3, 9) and from brains of C506M3 scrapie (lanes 1, 11) or classical BSE (lanes 2, 10) controls are shown for comparison. C - PrP res from spleens of mice infected with H-BSE at first passage (lanes 2, 4)(isolates 03-1928 and 03-2095), second passage in a mouse with “C-BSE like” PrP res (lane 6) and third passage from mice inoculated with a brain homogenate with “C-BSE like” PrP res (lanes 8, 10). PrP res from spleens of classical BSE (lanes 3, 5, 7, 9) and C506M3 (lanes 1, 11) controls are shown. Panels A and B were revealed by monoclonal antibody Sha31 and panel C by HRP-labelled SAF84 antibody Bars to the left indicate the 29.0 and 20.1 kDa marker positions in panels A and B or the 20.1 and 14.3 kDa marker positions in panels C and D.

Article Snippet: PrP res or PrP d were detected using the monoclonal antibodies anti-PrP Sha31 (1/10 from kit TeSeE sheep/goat Biorad), SAF84 (500 ng/ml)(SPI-Bio, France) or 12B2 (340 ng/ml) against the 144-WEDRYYRE-151, 163-RPVDQY-168 and 88-WGQGG-92 murine PrP sequences respectively.

Techniques: Comparison, Infection, Marker

Journal: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 1 PrPres western blot profiles in original E200K gCJD isolates and E200K gCJD-inoc- ulated human PrP-expressing mice. Western blot analysis of proteinase K-resistant prion protein (PrPres) profiles was performed on brain homogen- ates from both the original E200K gCJD patient isolates and transgenic mice expressing human PrP with Met129 (tgMet) or Val129 (tgVal) at codon 129. Mice were intracerebrally inoculated with 20 µL of 10% brain homogenate from E200K gCJD patients (n = 6 per group). Two serial passages were carried out in each mouse line (details in Table 1). The PrPres isoform (type 1 or type 2) was identified via SDS-PAGE and Western blot using the anti-PrP monoclonal antibody Sha31 (epitope YEDRYYRE). To control for PrPres isoform, MM1 sCJD (type 1) and VV2 sCJD (type 2) isolates were included in each gel. The PrPres isoform results for each passage and mouse line are summarized in Table 1

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Expressing, Transgenic Assay, SDS Page, Control

Journal: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 3 PrPres western blot in the brains of tgMet mice inoculated with brain and peripheral tissues from E200K gCJD Patients. Western blot analysis of proteinase K-resistant prion protein (PrPres) was conducted on brain homogenates from transgenic mice expressing human PrP with Met129 (tgMet) after two serial intracerebral passages (n = 6 per group). The mice were inoculated with 20µL of a 10% homogenate prepared from brain or peripheral tissues of E200K gCJD patients (refer to Tables 1 and 2). PrPres was detected using the anti-PrP mon- oclonal antibody Sha31, targeting the YEDRYYRE epitope. For com- parison, MM1 sCJD (type 1) and VV2 sCJD (type 2) prion isolates were included as controls. PrPres isoform identification results for each passage are summarized in Table 2

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Transgenic Assay, Expressing

Journal: Prion

Article Title: Identification of H-type BSE in Portugal

doi: 10.1080/19336896.2014.997615

Figure Lengend Snippet: Phenotypical features of the first Portuguese H-BSE. (a) PrPres immunolabelling in the reticular formation of the brainstem: linear pattern (arrow) and the predominant intraglial pattern as single granules in the white matter (arrowheads) (b). The box in picture (a) outlines the area shown in picture (b) at higher magnification. Specific PrPres signal was visualized with Vectastain–peroxidase, DAB (Vector). All sections were counterstained with Mayer's haematoxylin. The magnifications used are indicated. (c) Western blot analysis of PrPres after proteinase K treatment using Sha31 and P4 immunolabelling: Sha31 shows a higher molecular mass migration (orange arrow) by comparison to C-BSE; with P4 there is immunolabelling in the Portuguese H-BSE (PT H-BSE) similar to that presented by the H-BSE control. Absence of signal in C-BSE and L-BSE. H-BSE, C-BSE and L-BSE controls from AHVLA proficiency testing. Scrapie represents classical scrapie control. M, molecular mass marker (kDa).

Article Snippet: The confirmation of the H-type BSE phenotype was carried out using the discriminatory molecular method with the C-terminal anti-PrP antibody Sha31 and the N- terminal anti-PrP monoclonal antibody P4 (aa 101–107 bovine PrP protein, 15 R-Biopharm, 0.1mg/ml) diluted 1/500 as recommended by the European Union Reference Laboratory (AHVLA Bio-Rad TeSeE- based Hybrid Western Blot method).

Techniques: Plasmid Preparation, Western Blot, Migration, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: The Scrapie Prevalence in a Goat Herd Is Underestimated by Using a Rapid Diagnostic Test

doi: 10.3389/fbioe.2020.00164

Figure Lengend Snippet: Western blot profile of digested brain samples from selected goats using two monoclonal antibodies. (A) Sha31 antibody. (B) P4 antibody. Lanes 1 and 10: molecular mass marker; lane 2: goat 2135 (clinical suspect, positive on brain by IHC and ELISA); lane 3: goat 2113, clinical suspect, positive on brain by IHC and ELISA); lane 4: goat 2078 (no clinical signs of scrapie, positive on brain by IHC, negative by ELISA); lane 5: goat 2117 (no clinical signs of scrapie, negative on brain by IHC and ELISA, positive on lymphoid tissue by IHC; scrapie profile difficult to discern with the picture contrast used); lane 6: goat 2102 (clinical suspect, negative on brain by ELISA and on all tissues by IHC); lane 7: caprine classical scrapie control (RSCRAP 17/00006, II 142 QQ 222 ); lane 8: bovine classical BSE control (RBSE 98/00291); lane 9: ovine classical scrapie control (PG1903/97).

Article Snippet: Primary antibodies were Sha31 (Bio-Rad Laboratories, directed against the 148-YEDRYYRE-155 epitope), which was prepared according to the manufacturer’s kit instructions, and the N-terminal antibody P4 (R-Biopharm Rhône Ltd., Glasgow, United Kingdom, directed against the 93-WGQGGSH-99 epitope), which was used in a dilution 1:5000 of a stock solution of 1 mg/ml.

Techniques: Western Blot, Marker, Enzyme-linked Immunosorbent Assay