Production of IgA monoclonal antibody against Shiga toxin binding subunits employing nasal-associated lymphoid tissue
Production of IgA monoclonal antibody against Shiga toxin binding subunits employing nasal-associated lymphoid tissue
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DOI：
10.1016/j.jim.2005.05.007
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发表时间：
2005-07-01
影响因子：
2.2
通讯作者：
Kurohane, K
中科院分区：
文献类型：
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作者：
Imai, Y;Ishikawa, T;Kurohane, K
我们从 BALB/c 小鼠的鼻相关淋巴组织 (NALT) 中建立了针对志贺毒素 1B 亚基 (Stx1B) 的 IgA 单克隆抗体 (mAb)。我们开发了一种改进的方案，其中交联的 Stx1B 与霍乱毒素一起鼻内给药。富集以这种方式免疫的小鼠的表面IgA阳性NALT淋巴细胞，然后与小鼠骨髓瘤细胞融合以产生杂交瘤细胞。检查杂交瘤培养物上清液，看看它们是否含有针对 Stx1B 的 IgA，以及它们是否可以抑制 Stx1B 的碳水化合物识别。为了后一个目的，我们制备了碳水化合物配体，其中球三糖存在于聚赖氨酸主链上。建立的 IgA mAb 与固定的 Stx1B 表现出饱和且剂量依赖性的结合。相反，碳水化合物配体与固定化 Stx1B 的结合受到 mAb 预处理的抑制。免疫印迹和 SDS-PAGE 分析显示 IgA 二聚体。 IgA mAb 抑制地高辛结合的 Stx1B 与伯基特淋巴瘤细胞系 Ramos 上展示的天然配体的结合。这些结果表明，上呼吸道粘膜免疫系统诱导位点的表面 IgA 阳性 B 细胞是生产针对 Stx1B 等弱免疫原性蛋白抗原的 IgA mAb 的有效来源。 (c) 2005 Elsevier B.V. 保留所有权利。
We established an IgA monoclonal antibody (mAb) against Shiga toxin 1B subunits (Stx1B) from mouse nasal-associated lymphoid tissues (NALT) of BALB/c mice. We have developed an improved protocol in which cross-linked Stx1B is intranasally administered together with cholera toxin. Surface IgA-positive NALT lymphocytes from mice immunized in this manner were enriched and then fused with mouse myeloma cells to produce hybridoma cells. Hybridoma culture supernatants were examined to see if they contain IgA against Stx1B and if they can inhibit carbohydrate recognition by Stx1B. For the latter purpose, we prepared carbohydrate ligands in which globotriose is present on the poly-lysine backbone. The established IgA mAb exhibited saturable and dose-dependent binding to the immobilized Stx1B. Inversely, the binding of the carbohydrate ligands to the immobilized Stx1B was inhibited by the mAb pretreatment. Immunoblotting and SDS-PAGE analysis revealed dimeric IgA. The IgA mAb inhibited the binding of digoxigenin-conjugated Stx1B to natural ligands displayed on a Burkitt's lymphoma cell line, Ramos. These results suggested that surface IgA-positive B cells in the inductive sites of the mucosal immune system in the upper respiratory tract are a potent source for producing IgA mAb against protein antigens with weak immunogenicity such as Stx1B. (c) 2005 Elsevier B.V. All rights reserved.