title: Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab')2 fragments of monoclonal antibodies.
abstract: 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')(2) fragment. 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')(2) fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry. .
authors: Department of Advanced Medicine and Development, BML Inc., Saitama, Japan.
authors: Ryutaro
authors: R
authors: Oba
authors: Koji
authors: K
authors: Orihara
authors: Tomoaki
authors: T
authors: Kumagai
authors: Hiroyuki
authors: H
authors: Hirai
authors: Kinya
authors: K
authors: Nagata
authors: Shuichi
authors: S
authors: Hamasaki
authors: Chuwa
authors: C
authors: Tei
authors: Nakamura
authors: N
authors: Masataka
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001424:Bacterial Infections / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism; D016207:Cytokines / Q000276:immunology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D016207:Cytokines; D007140:Immunoglobulin Fab Fragments; D017452:Receptors, IgG; D051195:Toll-Like Receptor 2