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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 histopathology.(A) Histoblot analysis. Cryosections of scrapie ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were blotted onto a PVDF membrane. The histoblot membranes were used without autoclaving or denaturing pretreatments. The PrP signal was detected by mAbs 6A12 and 8D5 in scrapie-affected but not unaffected mouse brain. MAb 31C6 reacted with PrP in scrapie-affected and unaffected mouse brain. MAb P2b showed no signal. (B) Immunohistochemical analysis. Cryosections from ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were fixed with acetone. Sections were incubated with primary antibodies without antigen retrieval. Immunostaining was performed using mAbs 31C6, 6A12, and 8D5. The bar represents 50 µm. MAb 31C6 demonstrated a weak PrP signal in unaffected and scrapie-affected mouse brain. In contrast, mAbs 6A12 and 8D5 demonstrated strong PrP signals in scrapie-affected mouse brain but not in unaffected brain.
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pone-0058013-g003: Immunoreactivity of mAbs 6A12 and 8D5 in histopathology.(A) Histoblot analysis. Cryosections of scrapie ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were blotted onto a PVDF membrane. The histoblot membranes were used without autoclaving or denaturing pretreatments. The PrP signal was detected by mAbs 6A12 and 8D5 in scrapie-affected but not unaffected mouse brain. MAb 31C6 reacted with PrP in scrapie-affected and unaffected mouse brain. MAb P2b showed no signal. (B) Immunohistochemical analysis. Cryosections from ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were fixed with acetone. Sections were incubated with primary antibodies without antigen retrieval. Immunostaining was performed using mAbs 31C6, 6A12, and 8D5. The bar represents 50 µm. MAb 31C6 demonstrated a weak PrP signal in unaffected and scrapie-affected mouse brain. In contrast, mAbs 6A12 and 8D5 demonstrated strong PrP signals in scrapie-affected mouse brain but not in unaffected brain.

Mentions: In histoblot analysis, pan-PrP mAb 31C6 reacted with PrP from unaffected and scrapie-affected mouse brains. MAbs 6A12 and 8D5 demonstrated positive immunoreactivity in brains from scrapie-affected mice brain but not from unaffected mice (Fig. 3A). In acetone-fixed tissue sections, mAb 31C6 reacted faintly with PrP from unaffected and scrapie-affected mice without antigen retrieval. MAbs 6A12 and 8D5 strongly reacted with PrP in scrapie-affected mice, but no signal was detected in unaffected mice (Fig. 3B). These results indicate that mAbs 6A12 and 8D5 specifically react with PrPScin situ.


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 histopathology.(A) Histoblot analysis. Cryosections of scrapie ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were blotted onto a PVDF membrane. The histoblot membranes were used without autoclaving or denaturing pretreatments. The PrP signal was detected by mAbs 6A12 and 8D5 in scrapie-affected but not unaffected mouse brain. MAb 31C6 reacted with PrP in scrapie-affected and unaffected mouse brain. MAb P2b showed no signal. (B) Immunohistochemical analysis. Cryosections from ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were fixed with acetone. Sections were incubated with primary antibodies without antigen retrieval. Immunostaining was performed using mAbs 31C6, 6A12, and 8D5. The bar represents 50 µm. MAb 31C6 demonstrated a weak PrP signal in unaffected and scrapie-affected mouse brain. In contrast, mAbs 6A12 and 8D5 demonstrated strong PrP signals in scrapie-affected mouse brain but not in unaffected brain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585212&req=5

pone-0058013-g003: Immunoreactivity of mAbs 6A12 and 8D5 in histopathology.(A) Histoblot analysis. Cryosections of scrapie ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were blotted onto a PVDF membrane. The histoblot membranes were used without autoclaving or denaturing pretreatments. The PrP signal was detected by mAbs 6A12 and 8D5 in scrapie-affected but not unaffected mouse brain. MAb 31C6 reacted with PrP in scrapie-affected and unaffected mouse brain. MAb P2b showed no signal. (B) Immunohistochemical analysis. Cryosections from ME7-affected (scrapie), unaffected, and PrP0/0 mouse brains were fixed with acetone. Sections were incubated with primary antibodies without antigen retrieval. Immunostaining was performed using mAbs 31C6, 6A12, and 8D5. The bar represents 50 µm. MAb 31C6 demonstrated a weak PrP signal in unaffected and scrapie-affected mouse brain. In contrast, mAbs 6A12 and 8D5 demonstrated strong PrP signals in scrapie-affected mouse brain but not in unaffected brain.
Mentions: In histoblot analysis, pan-PrP mAb 31C6 reacted with PrP from unaffected and scrapie-affected mouse brains. MAbs 6A12 and 8D5 demonstrated positive immunoreactivity in brains from scrapie-affected mice brain but not from unaffected mice (Fig. 3A). In acetone-fixed tissue sections, mAb 31C6 reacted faintly with PrP from unaffected and scrapie-affected mice without antigen retrieval. MAbs 6A12 and 8D5 strongly reacted with PrP in scrapie-affected mice, but no signal was detected in unaffected mice (Fig. 3B). These results indicate that mAbs 6A12 and 8D5 specifically react with PrPScin situ.

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