The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

Magdalini Polymenidou,Rita Moos,Martin Vey,Yong-Zhong Shi,Simone Hornemann,Iva Hafner-Bratkovič,Adriano Aguzzi,Christina Sigurdson,Michael N G James,Kurt Wuthrich,Roman Jerala,Mike Scott,Bill Yajima,Anne Bellon,Nat Kav,Graciela Garen

doi:10.1371/journal.pone.0003872

Abstract

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

Highlights

The first anti-PrP antisera were first reported in 1984 [1], generation of high affinity monoclonal anti-PrP antibodies has been hindered for a long time by the fact that wild type mice are immunotolerant against PrP [2,3,4]
A high level of PrP sequence conservation among species [8] limited the possibility of raising antibodies against species-specific epitopes and only a handful of high affinity antibodies were produced in wild type mice
Further experiments revealed that while these antibodies preferentially bind to mono and unglycosylated PrP, they bind to diglycosylated molecules with reduced binding strength

Summary

Introduction

The first anti-PrP antisera were first reported in 1984 [1], generation of high affinity monoclonal anti-PrP antibodies has been hindered for a long time by the fact that wild type mice are immunotolerant against PrP [2,3,4]. A high level of PrP sequence conservation among species [8] limited the possibility of raising antibodies against species-specific epitopes and only a handful of high affinity antibodies were produced in wild type mice. Many of the currently available anti-PrP antibodies have specificities directed against the central and C-terminal part of the molecule. This results in part from the choice of antigen (purified PrP27-30) in most previous attempts to generate anti-PrP antibodies, from which the flexible unstructured N-terminal part of PrP is excluded

Methods

Results

Conclusion