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The N-terminal sequence of prion protein consists an epitope specific to the abnormal isoform of prion protein (PrP(Sc)).

Masujin K, Kaku-Ushiki Y, Miwa R, Okada H, Shimizu Y, Kasai K, Matsuura Y, Yokoyama T - PLoS ONE (2013)

Bottom Line: Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively.This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc).We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

View Article: PubMed Central - PubMed

Affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.

ABSTRACT
The conformation of abnormal prion protein (PrP(Sc)) differs from that of cellular prion protein (PrP(C)), but the precise characteristics of PrP(Sc) remain to be elucidated. To clarify the properties of native PrP(Sc), we attempted to generate novel PrP(Sc)-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP(Sc) purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP(Sc) from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP(C) from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively. This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc). We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

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Immunoreactivity of mAbs 6A12 and 8D5 in immunoprecipitation assays.Brain homogenate was incubated with mAb and immunoprecipitated with protein G-coupled magnetic beads. Immunoprecipitated sample was subjected to western blotting and detected by using HRP-conjugated mAb T2. MAbs 6H4 and SAF32 were used as positive controls. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as a negative control. The brain homogenates (250 µL) of BSE-affected [0.1–0.01% (w/v)], unaffected [1–0.1% (w/v)], and PrP0/0 [1% (w/v)] mice were used. Molecular weight markers are shown on the left (kDa). The concentration of all mAbs for the immunoprecipitation assay was 1 µg/mL. MAbs 6A12 and 8D5 selectivity reacted to PrP from prion-affected individuals.
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pone-0058013-g001: Immunoreactivity of mAbs 6A12 and 8D5 in immunoprecipitation assays.Brain homogenate was incubated with mAb and immunoprecipitated with protein G-coupled magnetic beads. Immunoprecipitated sample was subjected to western blotting and detected by using HRP-conjugated mAb T2. MAbs 6H4 and SAF32 were used as positive controls. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as a negative control. The brain homogenates (250 µL) of BSE-affected [0.1–0.01% (w/v)], unaffected [1–0.1% (w/v)], and PrP0/0 [1% (w/v)] mice were used. Molecular weight markers are shown on the left (kDa). The concentration of all mAbs for the immunoprecipitation assay was 1 µg/mL. MAbs 6A12 and 8D5 selectivity reacted to PrP from prion-affected individuals.

Mentions: We performed immunoprecipitation assays to confirm the immunoreactivity of the newly generated mAbs to native PrPSc. The mAbs 6A12 and 8D5 precipitated PrP from the brains of BSE-affected mice [0.1–0.01% (w/v)], but not from those of unaffected mice [1–0.1% (w/v)] (Fig. 1). In contrast, mAb 6H4 strongly recognized PrP from unaffected mice. One mAb, SAF32, which recognizes the octapeptide repeat region as an epitope, reacted equally to PrP in normal and BSE-affected mice (Fig. 1). These results suggest that mAbs 6A12 and 8D5 selectively immunoprecipitated PrPSc from BSE-affected mice.


The N-terminal sequence of prion protein consists an epitope specific to the abnormal isoform of prion protein (PrP(Sc)).

Masujin K, Kaku-Ushiki Y, Miwa R, Okada H, Shimizu Y, Kasai K, Matsuura Y, Yokoyama T - PLoS ONE (2013)

Immunoreactivity of mAbs 6A12 and 8D5 in immunoprecipitation assays.Brain homogenate was incubated with mAb and immunoprecipitated with protein G-coupled magnetic beads. Immunoprecipitated sample was subjected to western blotting and detected by using HRP-conjugated mAb T2. MAbs 6H4 and SAF32 were used as positive controls. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as a negative control. The brain homogenates (250 µL) of BSE-affected [0.1–0.01% (w/v)], unaffected [1–0.1% (w/v)], and PrP0/0 [1% (w/v)] mice were used. Molecular weight markers are shown on the left (kDa). The concentration of all mAbs for the immunoprecipitation assay was 1 µg/mL. MAbs 6A12 and 8D5 selectivity reacted to PrP from prion-affected individuals.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585212&req=5

pone-0058013-g001: Immunoreactivity of mAbs 6A12 and 8D5 in immunoprecipitation assays.Brain homogenate was incubated with mAb and immunoprecipitated with protein G-coupled magnetic beads. Immunoprecipitated sample was subjected to western blotting and detected by using HRP-conjugated mAb T2. MAbs 6H4 and SAF32 were used as positive controls. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as a negative control. The brain homogenates (250 µL) of BSE-affected [0.1–0.01% (w/v)], unaffected [1–0.1% (w/v)], and PrP0/0 [1% (w/v)] mice were used. Molecular weight markers are shown on the left (kDa). The concentration of all mAbs for the immunoprecipitation assay was 1 µg/mL. MAbs 6A12 and 8D5 selectivity reacted to PrP from prion-affected individuals.
Mentions: We performed immunoprecipitation assays to confirm the immunoreactivity of the newly generated mAbs to native PrPSc. The mAbs 6A12 and 8D5 precipitated PrP from the brains of BSE-affected mice [0.1–0.01% (w/v)], but not from those of unaffected mice [1–0.1% (w/v)] (Fig. 1). In contrast, mAb 6H4 strongly recognized PrP from unaffected mice. One mAb, SAF32, which recognizes the octapeptide repeat region as an epitope, reacted equally to PrP in normal and BSE-affected mice (Fig. 1). These results suggest that mAbs 6A12 and 8D5 selectively immunoprecipitated PrPSc from BSE-affected mice.

Bottom Line: Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively.This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc).We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

View Article: PubMed Central - PubMed

Affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.

ABSTRACT
The conformation of abnormal prion protein (PrP(Sc)) differs from that of cellular prion protein (PrP(C)), but the precise characteristics of PrP(Sc) remain to be elucidated. To clarify the properties of native PrP(Sc), we attempted to generate novel PrP(Sc)-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP(Sc) purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP(Sc) from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP(C) from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively. This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc). We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

Show MeSH
Related in: MedlinePlus