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 Fcc receptors (FccRs) 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 0 ) 2 ] fragments to avoid nonantigen-specific binding to FccRs. 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 0 ) 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 0 ) 2 fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry. ' We developed previously a new quantitative flow cytometry (FCM) analysis system for TLR2 using poly-styrene beads, which were covalently bound with recombinant TLR2 (rTLR2) as calibrators (6). The system allowed us to conduct longitudinal time series and follow-up studies about the modulation of TLR2 expression levels on monocytes without being influenced by fluctuations in the performance of antibody reagents, photomultiplier sensitivity, and other factors in FCM. Our previous studies using this assay system suggested that TLR2 level in monocytes seems to reflect the clinical host's immune status in patients with infection (6-8).

Changes in CD163+, CD11b+, and CCR2+ peripheral monocytes relate to Parkinson’s disease and cognition

“…Previous results reported for TLRs in other PD cohorts are conflicting (34, 35, 37). Several factors might account for this: namely, the constitutively low TLR4 surface expression and its tightly regulated endocytic transport, which may be influenced during blood purification; the age-dependent TLR downregulation (61); and the unspecific binding of non-recombinant TLR2 antibodies (62). Altogether, this makes TLRs difficult markers to compare across studies.…”