Prion protein-specific antibodies that detect multiple TSE agents with high sensitivity.

Sandra Mccutcheon,Boon Chin Tan,Lorenzo Gonzalez,E Fiona Houston,Jan P M Langeveld,Andrew C Gill,Stuart Martin,A Richard Alejo Blanco,Nora Hunter,Lauren Campbell,James Alibhai,Christopher De Wolf

doi:10.1371/journal.pone.0091143

Sandra Mccutcheon, Boon Chin Tan + Show 10 more

Open Access

https://doi.org/10.1371/journal.pone.0091143

Copy DOI

Journal: PloS one	Publication Date: Mar 7, 2014
Citations: 88	License type: CC BY 4.0

Affiliation: Roslin Institute, Animal and Plant Health Agency

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.

Highlights

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that affect both animals and man and include bovine spongiform encephalopathy (BSE), scrapie and variant Creutzfeldt-Jakob disease
TSE infection is accompanied by the molecular conversion of a host-encoded glycoprotein, PrPC, into a diseasedassociated aggregated isoform (PrPSc, [1]); this isoform is partially resistant to proteolytic degradation and accumulates in the brain of infected individuals and often in peripheral tissues prior to neuroinvasion
Following treatment with proteinase K (PK), different forms of PrP, which vary in relative molecular mass and result directly from differential cleavage events that are related to the strain of TSE agent, can be observed in animals and humans using both Western blotting and immunohistochemical approaches in an antibody-dependent manner [2– 6]

Summary

Introduction

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that affect both animals and man and include bovine spongiform encephalopathy (BSE), scrapie and variant Creutzfeldt-Jakob disease (vCJD). TSE infection is accompanied by the molecular conversion of a host-encoded glycoprotein, PrPC, into a diseasedassociated aggregated isoform (PrPSc, [1]); this isoform is partially resistant to proteolytic degradation and accumulates in the brain of infected individuals and often in peripheral tissues prior to neuroinvasion. Both PrPC and PrPSc can be differentially glycosylated (at asparagine residues 184 and 200, ovine sequence), possess a single disulphide bond and carry a C-terminal glycosylphosphatidylinositol anchor; whilst PrPC and PrPSc have the same primary structure, they differ both in their biochemical properties (such as solubility in detergents, resistance to proteolytic cleavage, denaturation with chaotropes i.e. guanidium) and secondary and tertiary structure. Amino acid variation between codons 106–112 (human numbering) strongly influences the binding of the antibody 3F4 [13,14]

Methods

Results

Conclusion