mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP Sc in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP C . mAb T2 showed an excellent inhibitory effect on PrP Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrPSc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP Sc accumulation.Key words anti-prion effect, monoclonal antibody, single-chain fragment variable region.Prion diseases, also called transmissible spongiform encephalopathies, are a group of neurodegenerative disorders, which includes BSE in cattle, CWD in deer and elk, scrapie in sheep and goats, and CJD in humans. The recent emergence of a new human prion disease, variant CJD, which is thought to result from consumption of BSEinfected materials by humans (1), is a major public health and safety issue (2, 3). The infectious agent, or prion, is mainly composed of PrP Sc , the detergent-insoluble and partially protease-resistant isoform of the host-encoded Anti-prion effect by scFv PrP Sc accumulation in prion-infected cell cultures, presumably by disrupting the PrP C -PrP Sc interaction (6, 7) and transgenic expression of anti-PrP mAb fragments prevents prion pathogenesis in mice (8). However, administration of mAbs has resulted in prevention of prion pathogenesis only when applied simultaneously, or shortly after, peripheral prion infection (9). This is probably due to poor diffusion of administered antibodies from vessels into tissues, particularly into the CNS. One study has demonstrated apoptosis of hippocampal and cerebellar neurons following intracerebral injection of mAbs reactive with PrP C (10), indicating that cross-linking or clustering PrP C by the anti-PrP antibody may trigger an abnormal signaling pathway. One possible means of delivering antiPrP antibodies efficiently into the CNS, and preventing undesirable adverse effects of such administration, is scFv antibodies, in which the polypeptides of antibody variable regions essential to antigen-binding are genetically linked through a polypeptide linker sequence (11-13). Because of its large size, a whole antibody molecule, with its relatively complex structure consisting of heavy and light polypeptide chains, is unsuitable for efficient gene manipulation with vectors. H...

The conformation of abnormal prion protein (PrPSc) differs from that of cellular prion protein (PrPC), but the precise characteristics of PrPSc remain to be elucidated. To clarify the properties of native PrPSc, we attempted to generate novel PrPSc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrPSc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrPSc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrPC 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 PrPSc-specific epitope exists in the N-terminal region of PrPSc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrPSc. We found that the ratio of proteinase K (PK)-sensitive PrPSc to PK-resistant PrPSc was constant throughout the disease time course.

ABSTRACT. The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues.

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