Journal: Acta Neuropathologica

Article Title: Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD

doi: 10.1007/s00401-022-02482-9

Figure Lengend Snippet: Biochemical characteristics of protease-resistant hCWD-prion protein of Wisc-1-inoculated tg650 mice. Western blot analysis of brain homogenates of tg650 mice inoculated with Wisc-1 isolate and digested with 200 µg/mL of PK using anti-PrP mAbs, from top to bottom, Sha31 (aa 145–152), 12F10 (aa 145–155), 9A2 (aa 102–104), and 12B2 (aa 93–97). A negative control, tg650 #3013, as well as a positive control (sCJD, MM1 subtype) were also included in the Western blot. Higher exposure for mAb 9A2 is also shown. The bands seen at 25 kDa are nonspecific bands that react with certain Abs

Article Snippet: PVDF membranes were blocked for 1 h in PBS-Tween (0.1%) containing skim milk powder (5%) and probed using anti-PrP monoclonal antibodies Sha31 (aa 145–152; 1:15,000; Cayman), 12F10 (aa 145–155; 1:200; Cayman), and 9A2 (aa 102–104) and 12B2 (aa 93–97; 1:1000; Wageningen Bioveterinary Research) overnight at 4 °C.

Techniques: Western Blot, Negative Control, Positive Control

Journal: Acta Neuropathologica

Article Title: Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD

doi: 10.1007/s00401-022-02482-9

Figure Lengend Snippet: Transmission of CWD-tg650 brain/spinal cord homogenates from mouse #327 to tg650 mice. Western blot analysis of brain homogenates from tg650 mouse (#3063; 2nd passage) inoculated with brain/spinal cord homogenates from tg650-Wisc-1 #327 (1st passage) and digested with 200 µg/mL of PK using anti-PrP mAbs, from top to bottom, Sha31 (aa 145–152), 12F10 (aa 145–155), 9A2 (aa 102–104), and 12B2 (aa 93–97). A negative control, tg650 #3011, as well as a positive control (sCJD) were included in the Western blot. Higher exposures for mAbs, 12F10 and 9A2, are also shown. The bands seen at 25 kDa are nonspecific bands that react with certain Abs

Article Snippet: PVDF membranes were blocked for 1 h in PBS-Tween (0.1%) containing skim milk powder (5%) and probed using anti-PrP monoclonal antibodies Sha31 (aa 145–152; 1:15,000; Cayman), 12F10 (aa 145–155; 1:200; Cayman), and 9A2 (aa 102–104) and 12B2 (aa 93–97; 1:1000; Wageningen Bioveterinary Research) overnight at 4 °C.

Techniques: Transmission Assay, Western Blot, Negative Control, Positive Control

Journal: PLoS Pathogens

Article Title: Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie

doi: 10.1371/journal.ppat.1005077

Figure Lengend Snippet: Fragments identified by mass spectrometry in the brains of scrapie-diseased tgOv rabbit and specific of the sheep sequence are colored. Bold: Sha31 anti-PrP epitope.

Article Snippet: Immunoblots revealed with the mAb Sha31 were quantified using the Alpha-Ease software (Alpha-Innotech, USA).

Techniques: Mass Spectrometry, Sequencing

Journal: PLoS Pathogens

Article Title: Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie

doi: 10.1371/journal.ppat.1005077

Figure Lengend Snippet: (a) PrP C electrophoretic pattern and level of expression in the brain of wild-type, sheep (VRQ allele), tgOv rabbit and tg338 mice. The amounts of material loaded are indicated. (b) Two-dimensional electrophoretic gel analysis of PrP C from wild-type and tgOv rabbit. The equivalent of 1mg of brain extract was used for comparison (Acidic side at left). Blots were probed with Sha31 anti-PrP antibody.

Article Snippet: Immunoblots revealed with the mAb Sha31 were quantified using the Alpha-Ease software (Alpha-Innotech, USA).

Techniques: Expressing, Comparison

Journal: PLoS Pathogens

Article Title: Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie

doi: 10.1371/journal.ppat.1005077

Figure Lengend Snippet: Midbrain sections from tgOv (a-b, e-f) and wild-type (c, g-i) rabbits challenged with LA21K fast scrapie prions and from a mock-infected tgOv rabbit (d, i-j). (a-d) PET blot analyses using monoclonal antibody Sha31 showed PrP res accumulation solely in LA21K fast challenged tgOv rabbits. (e-j) Hematoxylin and eosin-stained section at the level of the thalamus (e, g, i) and hippocampus (f, h, j) showing vacuolation, predominantly in the thalamus (e) and to a lesser extend in the hippocampus (f) of the LA21K fast challenged tgOv rabbits, but none in the control animals (g-j). Scale bar: 100 μm.

Article Snippet: Immunoblots revealed with the mAb Sha31 were quantified using the Alpha-Ease software (Alpha-Innotech, USA).

Techniques: Infection, Staining, Control

Journal: Emerging Infectious Diseases

Article Title: Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway

doi: 10.3201/eid1301.060393

Figure Lengend Snippet: Western blot profiles of PrPres in an atypical scrapie isolate (lane 2) detected by using N-terminal (4F2, P4), central (Sha31), or C-terminal (99/97.6.1) monoclonal antibodies. Molecular weight (MW) standard (lane 1). Immunoreactivities obtained with each antibody on 10 different atypical scrapie isolates are indicated (+, strong, ±, low, –, absent).

Article Snippet: Different monoclonal antibodies (MAbs) were used for detection of PrPres fragments: Sha 31 (4 μg/mL in PBST), P4 (1/2,500 in PBST) (R-Biopharm, Saint-Didier Au Mont d’Or, France), 4F2 (1/2,500 in PBST), and 99/97.6.1 (1/2,500 in PBST).

Techniques: Western Blot, Molecular Weight

Journal: Emerging Infectious Diseases

Article Title: Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway

doi: 10.3201/eid1301.060393

Figure Lengend Snippet: A) Schematic representation of ovine PrPc with location of epitopes recognized by the monoclonal antibodies used during the study and approaching sizes of PrPres fragments in atypical scrapie and Nor98 isolates. Theoretical apparent MWs of PrP fragments were calculated, by using those of each amino acid included in the known ARQ sheep PrP sequence, according to Sambrook and Russell . B) Interpretation of PrPres Western blot (WB) profiles in atypical scrapie and Nor98 isolates. Theoretical WB profile shows the expected apparent molecular masses of glycosylated PrP forms estimated by addition of 3.8 (*, monoglycosylated) or 7.9 (**, diglycosylated) kDa to the apparent molecular masses of A and B PrPres forms observed after PNGaseF deglycosylation. Values of 3.8 and 7.9 kDa were estimated from comparisons of glycosylated and unglycosylated forms in a classic scrapie isolate. The Sha31 WB profile included the mean apparent MWs assessed from highest resolution WB analysis (n = 32) and showed 2 separate peaks of maximal intensity in pictograms of signal intensities of bands II and III (19 sheep scrapie isolates).

Article Snippet: Different monoclonal antibodies (MAbs) were used for detection of PrPres fragments: Sha 31 (4 μg/mL in PBST), P4 (1/2,500 in PBST) (R-Biopharm, Saint-Didier Au Mont d’Or, France), 4F2 (1/2,500 in PBST), and 99/97.6.1 (1/2,500 in PBST).

Techniques: Sequencing, Western Blot