A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies

Vranac, Tanja; Hartman, Katrina; Popović, Mara; Venturini, Anja Colja; Žerovnik, Eva; Šerbec, Vladka Čurin

doi:10.1016/j.peptides.2006.05.026

Cited by 14 publications

(19 citation statements)

References 34 publications

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“…The further specific monoclonal antibodies used were: the EB8 anti-PrP monoclonal antibody that recognizes an N-terminal epitope (between residues 20 and 40 of human PrP; our unpublished data); and the C7/5 anti-PrP monoclonal antibody that recognizes a C-terminal epitope (residues 214 to 226 of human PrP [36]). These primary antibodies were revealed using horseradish-peroxidase-conjugated anti-mouse antibodies (Amersham) as the secondary antibodies.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

et al. 2012

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BackgroundAn R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrPSc) from different species (human, bovine, deer and mouse).Methodology/Principal FindingsDegradation of the PrPSc isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrPSc were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrPSc, the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58°C and 99°C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl2 were 105°C ±0.5°C and pH 6.5±0.2, respectively.Conclusions/SignificanceOur study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrPSc.

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Section: Methodsmentioning

confidence: 99%

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

et al. 2012

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“…Conformationally highly sensitive monoclonal antibodies that distinguish between PrP C and PrP Sc isoforms have been obtained in our group by immunizing BALB/c mice with a synthetic peptide from human PrP bound to a carrier molecule [23,24]. In the present study we show that it is possible to obtain a specific immune response against PrP even by immunizing wild-type mice expressing normal PrP with chemically unaltered recBoPrP.…”

Section: Introductionmentioning

confidence: 51%

“…The antibody titres were determined by enzyme-linked immunosorbent assay (ELISA) and the mouse with the highest titre was sacrificed. mAbs were prepared as described by Vranac et al [24].…”

Section: Immunization Of Balb/c Mice and The Generation Of Mabmentioning

confidence: 99%

Identification of an epitope on the recombinant bovine PrP that is able to elicit a prominent immune response in wild-type mice

Černilec¹,

Vranac²,

Hafner-Bratkovič³

et al. 2007

Immunology Letters

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“…Since the first successful production of high-affinity anti-prion protein (PrP) antibodies in PrP-knockout mice [12], several mAbs against PrP have been produced. However, only a few mAbs capable to distinguish PrP Sc form PrP C have beed reported [13], [14], [15], [16], [17], [18]. One of them is mAb V5B2, prepared against the C-terminal peptide P1 of the human PrP [13].…”

Section: Introductionmentioning

confidence: 99%

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

et al. 2011

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Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.

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A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies

Cited by 14 publications

References 34 publications

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

Identification of an epitope on the recombinant bovine PrP that is able to elicit a prominent immune response in wild-type mice

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

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