Development of sensitive diagnostic methods for transmissible spongiform encephalopathies.

开发传染性海绵状脑病的敏感诊断方法。

• 批准号: 07556068
• 负责人: SHINAGAWA Morikazu
• 金额: $ 7.55万
• 依托单位: Obihiro University of Agricutture and Veterinary Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556068/
• 关键词: Transmissible spongiform encephalopathies; Preclinical diagnosis; ELISA; ELISA法; 早期診断; ウエスタンブロット

In order to increase the sensitivity for detection of prion protein, the method to prepare samples from tissues for Western blotting was reformed. In mouse scrapie model, prion protein could be detected in the samples prepared from the spleen 1 week after intraperitoneal infection by the new method. It is to be desirable that more convenient method than Western blotting to examine many samples for the presence of prion protein. We developed an ELISA whose sensitivity was about two times higher than that of Western blotting using mouse model. We first confirmed that this ELISA method could be used for sheep scrapie samples, then applied the ELISA method for near 300 sheep samples obtained from slaughter house in Hokkaido.We are developing the method for preparing samples to detect prion protein in contaminated collagen. We found the ways to reduce viscosity of collagen solution by enzymes and to concentrate prion from large volume of the enzyme treated collagen solution.We also examined polymorphisms of sheep prion gene in relation to sheep with scrapie, expression of prion protein in various sheep organs, and polymorphisms of sheep apolipoprotein E gene as a risk factor of scrapie.

为了提高朊病毒蛋白检测的灵敏度，对组织样品的制备方法进行了改进。在小鼠瘙痒病模型中，腹腔感染后1周脾脏标本均可检测到朊病毒蛋白。希望有一种比免疫印迹法更方便的方法来检测许多样品中朊蛋白的存在。我们开发了一种灵敏度比小鼠免疫印迹法高2倍的酶联免疫吸附试验。本研究首先证实该方法可用于羊痒病样品，然后将该方法应用于北海道屠宰场近300只羊样品。我们正在开发一种检测被污染胶原蛋白中朊蛋白的样品制备方法。我们找到了用酶降低胶原溶液粘度和从经酶处理的大体积胶原溶液中浓缩朊病毒的方法。我们还研究了羊朊病毒基因多态性与羊瘙痒病的关系，朊病毒蛋白在羊各器官中的表达，以及羊载脂蛋白E基因多态性作为羊瘙痒病的危险因素。

项目成果

Horiuchi, M.et.al.: "A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep." J.Gen.Virol.76. 2583-2587 (1995)

Horiuchi, M.et.al.：“细胞形式的朊病毒蛋白 (PrPC) 存在于绵羊的许多非神经元组织中。”

Komatsu, Y.Horiuchi, M., Ishiguro, N., Matsui, T.and Shinagawa, M.: "Characterization of the sheep apolipoprotein E (Apo E) gene and allelic variations of the ApoE vgene in scrapie Suffolk sheep." Gene.(in press). (1998)

Komatsu, Y.Horiuchi, M.、Ishiguro, N.、Matsui, T. 和 Shinakawa, M.：“绵羊载脂蛋白 E (Apo E) 基因的特征以及瘙痒病萨福克羊中 ApoE v 基因的等位基因变异。”

Ikeda, T.et.al.: "Amino acid polymorphisms of PrP with reference to onset of scrapie in sSuffolk and Corriedale sheep in Japan." J.Gen.Virol.76. 2577-2581 (1995)

Ikeda, T.et.al.：“PrP 的氨基酸多态性与日本 sSuffolk 和 Corriedale 羊瘙痒症的发病有关。”

Horiuchi, M., Yamazaki, N., Ikeda, T., Ishiguro, N., and Shinagawa, M.: "A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep." J.Gen. Virol.76. 2583-2587 (1995)

Horiuchi, M.、Yamazaki, N.、Ikeda, T.、Ishiguro, N. 和 Shinakawa, M.：“朊病毒蛋白 (PrPC) 的细胞形式存在于绵羊的许多非神经元组织中。”

Grathwohl,K-U.et.al.: "Sensitive enzyme-Iinked immunosorbent assy for detection of PrPSc in crude tissue extracts from scrapie affected mice." J.Virol.Methods. 64. 205-216 (1997)

Grathwohl,K-U.et.al.：“用于检测瘙痒病小鼠粗组织提取物中 PrPSc 的灵敏酶联免疫吸附剂。”