Selective and Efficient Immunoprecipitation of the Disease-associated Form of the Prion Protein Can Be Mediated by Nonspecific Interactions between Monoclonal Antibodies and Scrapie-associated Fibrils

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

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Comparison of Three Monoclonal Antibodies for use in Immunohistochemical Detection of Bovine Spongiform Encephalopathy Protease-Resistant Prion Protein

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870601800117 ◽

2006 ◽

Vol 18 (1) ◽

pp. 106-109 ◽

Cited By ~ 3

Author(s):

Francisco J. Salguero ◽

Fayna Díaz-San Segundo ◽

Alejandro Brun ◽

María J. Cano ◽

Juan M. Torres

Keyword(s):

Monoclonal Antibodies ◽

Bovine Spongiform Encephalopathy ◽

Prion Protein ◽

Spongiform Encephalopathy ◽

Immunohistochemical Detection

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Mapping the Prion Protein Using Recombinant Antibodies

Journal of Virology ◽

10.1128/jvi.72.11.9413-9418.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 9413-9418 ◽

Cited By ~ 150

Author(s):

R. Anthony Williamson ◽

David Peretz ◽

Clemencia Pinilla ◽

Hadyn Ball ◽

Raiza B. Bastidas ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Surface ◽

Prion Protein ◽

Disease Process ◽

Cellular Prion Protein ◽

Epitope Region ◽

Molecular Events ◽

C Terminus ◽

Phage Display Libraries ◽

Recombinant Molecule

ABSTRACT The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPCbut absent from PrPSc.

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PrP glycoforms are associated in a strain-specific ratio in native PrPSc

Journal of General Virology ◽

10.1099/vir.0.80375-0 ◽

2005 ◽

Vol 86 (9) ◽

pp. 2635-2644 ◽

Cited By ~ 45

Author(s):

Azadeh Khalili-Shirazi ◽

Linda Summers ◽

Jacqueline Linehan ◽

Gary Mallinson ◽

David Anstee ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Prion Protein ◽

Prion Disease ◽

Prion Diseases ◽

Cellular Prion Protein ◽

Normal Brain ◽

Western Blots ◽

Prion Strains ◽

Specific Manner ◽

First Time

Prion diseases involve conversion of host-encoded cellular prion protein (PrPC) to a disease-related isoform (PrPSc). Using recombinant human β-PrP, a panel of monoclonal antibodies was produced that efficiently immunoprecipitated native PrPSc and recognized epitopes between residues 93–105, indicating for the first time that this region is exposed in both human vCJD and mouse RML prions. In contrast, monoclonal antibodies raised to human α-PrP were more efficient in immunoprecipitating PrPC than PrPSc, and some of them could also distinguish between different PrP glycoforms. Using these monoclonal antibodies, the physical association of PrP glycoforms was studied in normal brain and in the brains of humans and mice with prion disease. It was shown that while PrPC glycoforms can be selectively immunoprecipitated, the differentially glycosylated molecules of native PrPSc are closely associated and always immunoprecipitate together. Furthermore, the ratio of glycoforms comprising immunoprecipitated native PrPSc from diverse prion strains was similar to those observed on denaturing Western blots. These studies are consistent with the view that the proportion of each glycoform incorporated into PrPSc is probably controlled in a strain-specific manner and that each PrPSc particle contains a mixture of glycoforms.

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