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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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Biochemical analysis of PrPSc precipitated with the newly generated mAbs.PrPSc was precipitated by mAbs 6A12 and 8D5 from brain homogenate [0.05% (w/v)] from BSE-affected mice. The precipitated PrPSc was treated with (+) or without (−) 50 µg/mL of PK for 1 h and then western blotted with mAb T2. PrP signals were detected by HRP-conjugated mAb T2. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as negative control. The total amount of PrP and PrPcore in the brain homogenates used in this experiment was detected by routine western blotting (WB). PK treatment decreased the signal intensity of PrP, however, the typical three bands were detected.
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pone-0058013-g005: Biochemical analysis of PrPSc precipitated with the newly generated mAbs.PrPSc was precipitated by mAbs 6A12 and 8D5 from brain homogenate [0.05% (w/v)] from BSE-affected mice. The precipitated PrPSc was treated with (+) or without (−) 50 µg/mL of PK for 1 h and then western blotted with mAb T2. PrP signals were detected by HRP-conjugated mAb T2. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as negative control. The total amount of PrP and PrPcore in the brain homogenates used in this experiment was detected by routine western blotting (WB). PK treatment decreased the signal intensity of PrP, however, the typical three bands were detected.

Mentions: To clarify the characteristics of the mAbs, immunoprecipitated PrPSc was treated with PK. To detect the PrP band in WB, we used mAb T2, which recognizes the PrPcore region. Without PK digestion, precipitated PrPSc from BSE-affected mice exhibited an intense signal and a molecular weight consistent with that of intact PrP. In contrast, with PK treatment, precipitated PrPSc from BSE-affected mice showed a shifted molecular weight consistent with that of the PrPcore, and its signal intensity was decreased (Fig. 5). These results suggest that mAbs 6A12 and 8D5 isolate intact PrPSc, including the PK-resistant isoform of PrPSc, from prion-affected individuals.


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)

Biochemical analysis of PrPSc precipitated with the newly generated mAbs.PrPSc was precipitated by mAbs 6A12 and 8D5 from brain homogenate [0.05% (w/v)] from BSE-affected mice. The precipitated PrPSc was treated with (+) or without (−) 50 µg/mL of PK for 1 h and then western blotted with mAb T2. PrP signals were detected by HRP-conjugated mAb T2. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as negative control. The total amount of PrP and PrPcore in the brain homogenates used in this experiment was detected by routine western blotting (WB). PK treatment decreased the signal intensity of PrP, however, the typical three bands were detected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585212&req=5

pone-0058013-g005: Biochemical analysis of PrPSc precipitated with the newly generated mAbs.PrPSc was precipitated by mAbs 6A12 and 8D5 from brain homogenate [0.05% (w/v)] from BSE-affected mice. The precipitated PrPSc was treated with (+) or without (−) 50 µg/mL of PK for 1 h and then western blotted with mAb T2. PrP signals were detected by HRP-conjugated mAb T2. MAb P2b, which is isotype-matched with mAbs 6A12 and 8D5, was used as negative control. The total amount of PrP and PrPcore in the brain homogenates used in this experiment was detected by routine western blotting (WB). PK treatment decreased the signal intensity of PrP, however, the typical three bands were detected.
Mentions: To clarify the characteristics of the mAbs, immunoprecipitated PrPSc was treated with PK. To detect the PrP band in WB, we used mAb T2, which recognizes the PrPcore region. Without PK digestion, precipitated PrPSc from BSE-affected mice exhibited an intense signal and a molecular weight consistent with that of intact PrP. In contrast, with PK treatment, precipitated PrPSc from BSE-affected mice showed a shifted molecular weight consistent with that of the PrPcore, and its signal intensity was decreased (Fig. 5). These results suggest that mAbs 6A12 and 8D5 isolate intact PrPSc, including the PK-resistant isoform of PrPSc, from prion-affected individuals.

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