Table 1.
ELISA immunoreactivity of mAbs to native and denatured PrPSc.
Figure 1.
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.
Figure 2.
Immunoreactivity of mAbs 6A12 and 8D5 with PrPSc from different prion strains and animal species.
(A) The brain homogenates [0.05% (w/v)] from ME7- and Chandler-affected mice were used. BSE: brain homogenate from BSE-affected mice. (B) Cross-reactivity of mAbs 6A12 and 8D5 with PrPSc from BSE and scrapie. Brain homogenates from unaffected [1% (w/v)] and affected [0.5% (w/v)] individuals were used. Total PrP in the brain homogenate was detected by routine western blotting (WB). BSE: brain homogenate from C-BSE-affected cattle; Scrapie: brain homogenate from scrapie-affected sheep. 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 cross-reacted with PrPSc from different prion strains and animal species.
Figure 3.
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.
Figure 4.
(A) The epitopes of 6A12 and 8D5 in the PrP amino acid sequence. The epitope positions of mAbs 6A12 and 8D5 are represented by boxes under the amino acid sequence of mouse PrP (residues 1–254) (Supplemental Fig. 2). The dashed line indicates the octapeptide repeat region. Sequence numbers are shown on the right. (B) Peptide competition assay. P31–39 and P41–47 are synthetic peptides. MAbs were pre-incubated with (P31–39 or P41–47) and without (−) synthetic peptide prior to use in immunoprecipitation. The brain homogenate [0.05% (w/v)] from BSE-affected mice was incubated with the antibody-peptide complex and immunoprecipitated with protein G-coupled magnetic beads. The immunoprecipitated PrP was western blotted and detected with HPR-conjugated mAb T2. Total PrP in the brain homogenate was detected by routine western blotting (WB). Molecular weight markers are shown on the left (kDa). P31–39 blocked the reactivity of mAb 8D5 to PrPSc, but not the reactivity of mAb 6A12 to PrPSc. Conversely, P41–47 blocked the reactivity of mAb 6A12 to PrPSc, while P31–39 did not.
Figure 5.
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.
Figure 6.
Dynamics of PrPSc in BSE-affected mice.
BSE-affected mice were killed at 0, 40, 80, 120, and 150 dpi (terminal stage). (A) PrP detection by routine western blotting in disease stages. Brain homogenate [0.05% (w/v)] from BSE-affected mice was treated with (+) or without (−) 50 µg/mL PK for 1 h followed by western blotting with mAb T2. (B) PrPSc detection by immunoprecipitation. PrPSc was precipitated by mAb 6A12 from the same samples as in (A). The precipitated PrPSc was treated with (+) or without (−) PK, and PrP signals were detected by HRP-conjugated mAb T2. PK (−) samples indicate the total amount of PrPSc, and PK (+) samples represent the amount of PrPcore. Molecular weights are shown on the left (kDa). (C) Quantification of PrPSc signals in (B). The band intensity relative to total PrPSc (%) at the terminal stage is shown. Black bar: PrPcore. White bar: total PrPSc. All values were calculated as the mean ± standard deviation of at least 3 independent experiments.