@misc{oai:niigata-u.repo.nii.ac.jp:00005341,
 author = {山本, 卓司},
 month = {Mar},
 note = {Prion diseases are neurodegenerative diseases of humans and other animals. Affected animals accumulate an abnormal prion protein isoform (PrPSc), which is generated by posttranslational modification of cellular prion protein (PrPC). Unlike PrPC, PrPSc has a large number of β-sheets. This structure is thought to cause aggregation of PrPSc, which exhibits relative resistance to digestion by proteinase K (PK). The moiety remaining after digestion by PK is detected as PrPcore. According to the “protein-only” hypothesis, PrPSc is believed to be the major, or only, component of the infectious agent, the prion. Because the conformation of PrPSc seems to determine disease phenotype, conformational discrimination of PrPSc is considered to be a critical issue. In 2nd chapter, in order to develop novel PrPSc-specific monoclonal antibodies (mAbs), we immunized PrP-deficient mice with PrPSc that was purified from scrapie-infected mouse brains (SMB). Then, we developed PrPSc-specific mAbs by immunizing mice against PrPSc with the intention of producing a direct probe for PrPSc. These mAbs detected PrPSc only from the SMB homogenate by immunoprecipitation, and they could recognize conformational differences in the PrPSc of several species. The mAbs were used for studying the conformational transition of PrPSc during interspecies transmission of prion diseases. In 3 rd chapter, we developed a novel disposable homogenizer, named BioMasher. BioMasher was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy (BSE) 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. Then, 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 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., In 4th chapter, we developed novel diagnosis sandwitch ELISA method. There have been used four diagnosis kits for BSE in Japan. But, in these diagnosis kits, to prepare a tissue sample for use in an ELISA, the purification and concentration step using organic solvent or salt followed by proteinase K digestion was thought to be indispensable. Then, we developed a new screening method to detect BSE. This method is advantageous because it has a simpler and safer protocol than commercial kits. Especially, a purification step was eliminated in the sample preparation by using BioMasher. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. MAbs to prion protein were created and used to construct a sensitive sandwich ELISA system (1F5-T2 ELISA assay). The sensitivity of this 1F5-T2 ELISA assay using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37℃ for 1 day, is 10- to 30-fold more sensitive than a commercial kit. It was also tested for the detection of ovine scrapie, 50 scrapie-positive ovine samples from the UK, and 54 scrapie-negative ovine samples from Japan. The sensitivity and specificity of this 1F5-T2 ELISA assay for ovine samples were 96% and 100%, respectively. The detection limit of the PrPSc of 1F5-T2 ELISA assay was examined using diluted scrapie-positive samples. The sensitivity of 1F5-T2 ELISA assay to ovine scrapie was 3–10 times superior to that of another commercial BSE diagnosis kit. Thus, the 1F5-T2 ELISA assay proved more effective for the detection of ovine scrapie., 学位の種類: 博士（農学）. 報告番号: 甲第3794号. 学位記番号: 新大院博（農）甲第125号. 学位授与年月日: 平成25年3月25日, 新大院博（農）甲第125号},
 title = {抗体によるプリオン病診断法に関する研究},
 year = {2013}
}