Prion Protein-Specific Antibodies that Detect Multiple TSE Agents with High Sensitivity

Abstract

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration

“…To block for endogenous peroxidase, slides were immersed in 1% H 2 O 2 /methanol before washing in running water for 5 min followed by PBS/1% BSA wash buffer for 5 min. Sections were subsequently incubated with Normal Goat Serum (Stratech) for 20 min before application of either BH1 ([55] used at 0.02 μg/mL) or 6H4 (Prionics used at 3 μg/mL) anti-PrP antibodies. The primary antibodies were incubated overnight before washing with PBS/1% BSA wash buffer.…”

A prion reduction filter does not completely remove endogenous prion infectivity from sheep blood

“…Each assay included known BSE‐positive and ‐negative control tissue sections to verify the sensitivity and specificity of the procedure. Immunohistochemical staining of rectal biopsy samples was performed as previously described, using the MoAb R145 …”

“…Proteinase K (PK)-resistant PrP Sc in brain and lymphoid tissues was detected using established Western blot methods, probing with the monoclonal antibody (MoAb) ROS-BC6 (0.5 mg/mL) as previously described. 17,54,55 Autoradiography film (Hyperfilm, GE Healthcare, Buckinghamshire, UK) was scanned and the digital image processed using a computer program (Adobe Photoshop, Adobe Systems, Inc., San Jose, CA). Image processing was restricted to cropping, preparation of composite figures, and annotation.…”

“…Image processing was restricted to cropping, preparation of composite figures, and annotation. Formalin-fixed sections of brain and lymphoid tissues were also analyzed for PrP d deposition by immunohistochemistry using MoAbs ROS-IH9 55 and BG4, as previously described. The MoAbs were applied and incubated overnight-ROS-IH9 (0.5 mg/mL at 48C) or BG4 (1.25 mg/ mL at ambient temperature).…”

Prion Protein-Specific Antibodies-Development, Modes of Action and Therapeutics Application

“…Series of conventional antibodies have been raised since then, with promising candidates able to cure PrP Sc in vitro with half maximal inhibitory concentration of PrP Sc (IC50) far below 1 μM [ 50 ]. The process of developing new panels of anti-PrP antibodies is still in progress [ 45 , 93 , 94 , 95 , 96 ]. A study aimed at characterizing the pharmacokinetic properties of anti-PrP antibodies with curing properties in vitro showed that their curing capacity in vivo is associated with intrinsic pharmacokinetic properties rather than their isotype, epitope or affinity [ 97 ].…”