Characterization and epitope mapping of two monoclonal antibodies against human CD99
Characterization and epitope mapping of two monoclonal antibodies against human CD99
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DOI：
10.1038/emm.2002.58
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发表时间：
2002-12-31
影响因子：
12.8
通讯作者：
Kim, TJ
中科院分区：
文献类型：
--
作者：
Gil, MC;Lee, MH;Kim, TJ
CD 99在单核细胞的渗出、T细胞分化和MHC分子的转运中起关键作用。据报道，激动性单克隆抗体与CD 99的结合可触发多因素事件，包括造血细胞分化期间的T细胞活化以及细胞-细胞粘附。在这项研究中，为了鉴定参与细胞事件的功能结构域，我们绘制了CD 99的表位，这些表位被两种激动性CD 99单克隆抗体DN 16和YG 32识别。使用GST与整个或部分CD 99的重组融合蛋白，我们发现这两种抗体与CD 99分子相互作用独立的糖部分。DN 16单抗检测到一个位于CD 99氨基末端的线性表位，而YG 32单抗则检测到另一个位于胞外区中心的线性表位。为了证实鉴定的CD 99表位实际上被两种mAb识别，我们使用表面等离子体共振分析显示mAb与含有相应表位的融合蛋白或合成肽之间存在物理相互作用。DN 16和YG 32 mAb对抗原的解离常数分别计算为1.27 x 10（-7）和7.08 x 10（-9）M。这些研究将有助于了解CD 99的功能结构域和后续的信号转导机制。
CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 x 10(-7) and 7.08 x 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.