Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab')(2) fragments of monoclonal antibodies
Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab')(2) fragments of monoclonal antibodies
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使用单克隆抗体 F(ab)(2) 片段重新评估单核细胞上 TLR2 的定量流式细胞术分析
DOI：
10.1002/cyto.a.21000
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
大場隆一郎
中科院分区：
文献类型：
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作者：
Lo Li-Wei;Satoshi Higa;et al.;大場隆一郎
在难治性感染患者中，需要监测严重程度和愈合过程的可靠标志物。单核细胞上的Toll样受体2（TLR 2）的表达水平是这样的候选者。在常规测定系统中，抗TLR 2 mAb的完整IgG（wIgG）形式与阻断非抗原特异性结合的对照IgG一起使用。但是，应考虑标记抗TLR 2 mAb和对照IgG之间对Fcγ受体（Fcγ R）的竞争反应。我们的目标是通过流式细胞术（FCM）精确定量单核细胞上TLR 2的表达水平。在本研究中，我们制备了抗TLR 2 mAb D45（IgG 2a）和D29（IgG 1）及其抗原结合片段[F（ab′）2]，以避免与Fcγ R的非抗原特异性结合。然后，我们通过使用这些mAb/片段和我们的使用重组TLR 2珠的校准系统来确定单核细胞上的TLR 2表达水平。PE标记的D45 wIgG与单核细胞的结合被未标记的D45 wIgG完全阻断，但未被未标记的D45 F（ab′）2片段阻断。尽管D29 wIgG与未刺激的单核细胞的非抗原特异性结合可忽略不计，但在白细胞介素10刺激的单核细胞中其增强。经证明，用对照IgG处理难以完全阻断D45和D29 wIgG的非抗原特异性结合。研究证明，使用缺乏抗TLR 2 mAb可结晶片段部分的荧光标记抗原结合区[如PE标记的F（ab′）2片段]对于流式细胞术中单核细胞上TLR 2水平的定量是必不可少的。© 2010国际细胞计数促进学会
In patients with refractory infections, reliable markers that monitor the severity and healing process are needed. The expression level of toll‐like receptor 2 (TLR2) on monocytes is such candidate. In the conventional assay system, the whole IgG (wIgG) form of anti‐TLR2 mAb has been used with control IgG, which blocks nonantigen‐specific bindings. However, the competitive reactions against Fcγ receptors (FcγRs) between labeled anti‐TLR2 mAbs and control IgG should be considered. Our goal was to precisely quantify TLR2 expression level on monocytes by flow cytometry (FCM). In this study, we prepared anti‐TLR2 mAbs, D45 (IgG2a), and D29 (IgG1), as well as their fragment antigen‐binding [F(ab′)2] fragments to avoid nonantigen‐specific binding to FcγRs. And then, we determined TLR2 expression levels on monocytes by using these mAbs/fragments and our calibration system using recombinant TLR2 beads. The binding of PE‐labeled D45 wIgG to monocytes was completely blocked with unlabeled D45 wIgG, but not with unlabeled D45 F(ab′)2fragment. Although the nonantigen‐specific binding of D29 wIgG to nonstimulated monocytes was negligible, it was enhanced in interleukin‐10‐stimulated monocytes. It proved difficult to completely block nonantigen‐specific binding of D45 and D29 wIgGs by treatment with control IgG. It was demonstrated that the use of fluorescent‐labeled antigen‐binding region lacking the fragment crystallizable portion of anti‐TLR2 mAb [such as the PE‐labeled F(ab′)2fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry. © 2010 International Society for Advancement of Cytometry