Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab′)2 fragments of monoclonal antibodies

摘要: 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. conventional assay system, whole IgG (wIgG) form anti-TLR2 mAb has been used control IgG, which blocks nonantigen-specific bindings. However, competitive reactions against Fcγ receptors (FcγRs) between labeled mAbs should be considered. Our goal was to precisely quantify TLR2 by flow cytometry (FCM). this study, we prepared mAbs, D45 (IgG2a), D29 (IgG1), as well their fragment antigen-binding [F(ab')(2) ] fragments avoid binding FcγRs. And then, determined levels using these mAbs/fragments our calibration system recombinant beads. PE-labeled wIgG completely blocked unlabeled wIgG, but not F(ab')(2) fragment. Although nonstimulated negligible, it enhanced in interleukin-10-stimulated monocytes. It proved difficult block wIgGs treatment IgG. demonstrated use fluorescent-labeled region lacking crystallizable portion [such fragment] indispensible for quantification cytometry. .