Development of functional IgA antibodies against carbohydrate recognition activity of Vero toxin and their application to treatment.

针对 Vero 毒素碳水化合物识别活性的功能性 IgA 抗体的开发及其在治疗中的应用。

• 批准号: 12680638
• 负责人: IMAI Yasuyuki
• 金额: $ 0.64万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680638/
• 关键词: O157; Vero toxin; mucosal immunity; Carbohydrate recognition; IgA; pathogenic E. coli; EHEC; germinal center

We investigated mucosal immune response against Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) toward the end of development of IgA monoclonal antibodies. As an antigen, carbohydrate recognition subunits (Stx-1B) were expressed and the proteins were purified until homogeneity. The activity was confirmed by binding to glycolipid Gb_3 and to CD77 positive Burkitt's lymphoma cell lines. We prepared globotriose-conjugated poly-lysine, and their binding to immobilized Stx-1B was measured by an ELISA format. Antiserum from mice that had parenterally been immunized with the Stx-1B specifically interfered with the ligand binding. This result suggests the usefulness of the assay as a screening method of blocking antibodies. We also immunized mice through mucosal route using Stx-1B with cholera toxin as a mucosal adjuvant. We observed antigen specific serum IgG and IgA as well as specific secreted IgA in feces and intestinal wash. We also detected antigen specific IgA-producing cells from intestinal lamina propria using ELISPOT assay and the specific IgA in the culture supernatant of lamina propria lymphocytes from immunized mice. We then examined the ability of Stx-1B as a probe to detect biological ,ligands using an immunohistochemical approach. The Stx-1B specifically bound to renal tubules and collecting ducts in mouse kidney frozen sections. This is consistent with the result of human tissues. On the other hand, germinal centers developed in mouse lymph nodes and Peyer's patches were not stained by Stx-1B. The results suggested that, unlike human immune system, Stx would not impede germinal center functions (affinity maturation and class switch) of mouse immune system. This is consistent with the results that Stx-1Bspecific IgA was produced after mucosal immunization.

我们研究了粘膜免疫反应对滋贺毒素（Stxs）产生的肠出血性大肠杆菌（EHEC）接近尾声的伊加单克隆抗体的发展。作为抗原，表达碳水化合物识别亚基（Stx-1B），并纯化蛋白质直至均一。通过与糖脂Gb_3和CD 77阳性伯基特淋巴瘤细胞系的结合证实了活性。我们制备了globotriose-共轭聚赖氨酸，并通过ELISA形式测定它们与固定化Stx-1B的结合。来自用Stx-1B胃肠外免疫的小鼠的抗血清特异性地干扰配体结合。该结果表明该测定法作为阻断抗体的筛选方法是有用的。我们还通过使用Stx-1B与霍乱毒素作为粘膜佐剂的粘膜途径免疫小鼠。我们观察了抗原特异性血清IgG和伊加以及粪便和肠洗液中的特异性分泌伊加。用ELISPOT法检测肠固有层抗原特异性IgA产生细胞和免疫小鼠固有层淋巴细胞培养上清中特异性伊加。然后，我们研究的能力，Stx-1B作为探针，检测生物，配体使用免疫组织化学方法。在小鼠肾脏冰冻切片中，Stx-1B特异性结合于肾小管和集合管。这与人体组织的结果一致。另一方面，小鼠淋巴结和派伊尔集合淋巴结中发育的生发中心未被Stx-1B染色。结果表明，与人类免疫系统不同，Stx不会阻碍小鼠免疫系统的生发中心功能（亲和力成熟和类转换）。这与粘膜免疫后产生Stx-1B特异性伊加的结果一致。

项目成果

今井康之: "基礎生化学実験法第5巻 脂質・糖質・複合糖質"東京化学同人. 333 (2000)

今井康幸：《基础生物化学实验方法第 5 卷：脂质、碳水化合物、复合碳水化合物》东京化学同人社 333（2000）。

今井康之(分担執筆): "基礎生化学実験法 第5巻 脂質・糖質・複合糖質"東京化学同人. 333 (2000)

Yasuyuki Imai（合著者）：《基础生物化学实验方法第 5 卷：脂质、碳水化合物和复合碳水化合物》东京化学同人社 333（2000）。

Sayuri Miyashita: "Development of recombinant B subunit of Shiga-like toxin 1 as a probe to detect carbohydrate ligands in immunochemical and flowcytometric application."Glycoconjugate Journal. 16・11. 697-705 (1999)

Sayuri Miyashita：“开发志贺样毒素 1 的重组 B 亚基作为免疫化学和流式细胞术应用中的碳水化合物配体检测。” 糖结合物杂志 16・11 (1999)。

Yasuyuki Imai: "Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits"J. Biochem.,. 130. 665-670 (2001)

Yasuyuki Imai：“多价碳水化合物配体与固定志贺样毒素 1 B 亚基的 pH 敏感结合的演示”J.

Yasuyuki Imai: "Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits"J. Biochem.. 130. 665-670 (2001)

Yasuyuki Imai：“多价碳水化合物配体与固定志贺样毒素 1 B 亚基的 pH 敏感结合的演示”J.