Generation of anti-human DEC205/CD205 monoclonal antibodies that recognize epitopes conserved in different mammals

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• 作者：Chae Gyu Park^a ; ^{parkc@rockefeller.edu} ; Anthony Rodriguez^a ; Hisashi Ueta^b ; Haekyung Lee^a ; Maggi Pack^a ; Kenjiro Matsuno^b ; Ralph M. Steinman^a ; ¹
• 关键词：CHO cells ; Chinese hamster ovary cells ; CR domain ; cysteine rich domain ; CRD ; carbohydrate recognition domain ; DCs ; dendritic cells ; FCS ; fetal calf serum ; FN domain ; fibronectin type II domain ; hDEC205 ; human DEC205 ; Ig ; immunoglobulin ; KO ; knockout ; mAb
• 刊名：Journal of Immunological Methods
• 出版年：2012
• 期刊代码：57_00221759
• 类别：imm
• 出版时间：30 March, 2012
• 卷：377
• 期：1-2
• 页码：15-22
• 文件大小：1539 K

摘要

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.