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

我们研究了肠出血性大肠杆菌(EHEC)产生的志贺毒素(Stxs)的粘膜免疫反应，直到IgA单抗研制结束。以糖识别亚基(STX-1B)为抗原进行表达，纯化蛋白直至均一。通过与糖脂GB_3和CD77阳性的Burkitt淋巴瘤细胞株的结合证实了其活性。我们制备了球三糖偶联的多赖氨酸，并用ELISA法测定了它们与固定化STX-1B的结合力。用STX-1B特异性肠外免疫的小鼠的抗血清干扰了配体的结合。这一结果表明，该试验作为一种筛选封闭抗体的方法是有用的。我们还用霍乱毒素作为粘膜佐剂的STX-1B通过黏膜途径免疫小鼠。观察血清中抗原特异性免疫球蛋白Ig G和免疫球蛋白A，以及粪便和肠道冲洗液中特异性分泌的免疫球蛋白A。用ELISPOT法检测肠道固有层抗原特异性IgA产生细胞和免疫小鼠固有层淋巴细胞培养上清液中特异性IgA。然后，我们用免疫组织化学的方法检测了STX-1B作为探针检测生物配体的能力。在小鼠肾脏冰冻切片中，STX-1B与肾小管和集合管特异性结合。这与人体组织的研究结果一致。而在小鼠的淋巴结和Peyer‘s小结中，生发中心未见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(亲和力成熟和类切换)。这与粘膜免疫后产生STX-1B特异性IgA的结果一致。

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.