Laboratory of Cellular Physiology and Immunology and Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, New York, NY 10065, USA.
J Immunol Methods. 2012 Mar 30;377(1-2):15-22. doi: 10.1016/j.jim.2011.12.009. Epub 2012 Jan 16.
DEC205/CD205 is a C-type multilectin receptor, expressed highly in dendritic cells (DCs). Previous efforts to generate anti-human DEC205 (anti-hDEC205) monoclonal antibodies (mAbs) from mice immunized with subdomain proteins of hDEC205 resulted in a few mAbs. Recently, we expressed and utilized a full-length extracellular domain protein of hDEC205 to successfully generate 5 strong anti-hDEC205 mAbs from mice. In this study, DEC205 knockout (KO) mice were immunized with this full-length extracellular domain protein of hDEC205. One of the 3 immunized DEC205 KO mice was chosen for the highest anti-hDEC205 titer by flow cytometric analysis of serum samples on CHO cells stably expressing hDEC205 (CHO/hDEC205 cells) and used for hybridoma fusion. From a single fusion, more than 400 anti-hDEC205 hybridomas were identified by flow cytometric screen with CHO/hDEC205 cells, and a total of 115 hybridomas secreting strong anti-hDEC205 mAb were saved and named HD1 through HD115. To characterize in detail, 10 HD mAbs were chosen for superior anti-hDEC205 reactivity and further subjected to cloning and purification. Interestingly, out of those 10 chosen anti-hDEC205 HD mAbs, 5 mAbs were also strongly reactive to mouse DEC205 while 8 mAbs were found to stain DEC205(+) DCs on monkey spleen sections. In addition, we also identified that HD83, one of the 10 chosen HD mAbs, stains DEC205(+) DCs in rat spleen and lymph node. Therefore, by immunizing DEC205 KO mice with a full-length extracellular domain protein of hDEC205, we generated a large number of strong anti-hDEC205 mAbs many of which are cross-species reactive and able to visualize DEC205(+) DCs in lymphoid tissues of other mammals.
DEC205/CD205 是一种 C 型多凝集素受体，在树突状细胞 (DC) 中高度表达。之前，人们从用 hDEC205 亚结构域蛋白免疫的小鼠中生成抗人 DEC205（抗-hDEC205）单克隆抗体（mAb）的尝试只得到了少数 mAb。最近，我们表达并利用了 hDEC205 的全长细胞外结构域蛋白，成功地从小鼠中生成了 5 种强抗-hDEC205 mAb。在这项研究中，我们用该全长 hDEC205 细胞外结构域蛋白免疫 DEC205 敲除 (KO) 小鼠。通过流式细胞术分析 CHO 细胞（CHO/hDEC205 细胞）上稳定表达的 hDEC205 血清样本，从 3 只免疫的 DEC205 KO 小鼠中选择了一只具有最高抗-hDEC205 效价的小鼠用于杂交瘤融合。通过流式细胞术筛选 CHO/hDEC205 细胞，从单次融合中鉴定出超过 400 个抗-hDEC205 杂交瘤，总共保存并命名为 HD1 至 HD115 的 115 个分泌强抗-hDEC205 mAb 的杂交瘤。为了详细表征，选择了 10 个 HD mAb 用于检测其对 hDEC205 的高反应性，并进一步进行了克隆和纯化。有趣的是，在这 10 个选择的抗-hDEC205 HD mAb 中，有 5 个 mAb 也强烈反应 mouse DEC205，而 8 个 mAb 被发现可以染色猴脾切片上的 DEC205(+) DCs。此外，我们还鉴定出，10 个选择的 HD mAb 之一的 HD83 可以染色大鼠脾和淋巴结中的 DEC205(+) DCs。因此，通过用 hDEC205 的全长细胞外结构域蛋白免疫 DEC205 KO 小鼠，我们生成了大量强抗-hDEC205 mAb，其中许多 mAb 具有跨物种反应性，能够在其他哺乳动物的淋巴组织中可视化 DEC205(+) DCs。
