PMID- 21432991
OWN - NLM
STAT- MEDLINE
DCOM- 20110701
LR  - 20191210
IS  - 1552-4930 (Electronic)
IS  - 1552-4922 (Linking)
VI  - 79
IP  - 4
DP  - 2011 Apr
TI  - Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using 
      F(ab')2 fragments of monoclonal antibodies.
PG  - 247-55
LID - 10.1002/cyto.a.21000 [doi]
AB  - 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 Fcgamma receptors (FcgammaRs) 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 
      FcgammaRs. 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. .
CI  - Copyright (c) 2010 International Society for Advancement of Cytometry.
FAU - Oba, Ryutaro
AU  - Oba R
AD  - Department of Advanced Medicine and Development, BML Inc., Saitama, Japan.
FAU - Orihara, Koji
AU  - Orihara K
FAU - Kumagai, Tomoaki
AU  - Kumagai T
FAU - Hirai, Hiroyuki
AU  - Hirai H
FAU - Nagata, Kinya
AU  - Nagata K
FAU - Hamasaki, Shuichi
AU  - Hamasaki S
FAU - Tei, Chuwa
AU  - Tei C
FAU - Masataka, Nakamura
AU  - Masataka N
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
DEP - 20101223
PL  - United States
TA  - Cytometry A
JT  - Cytometry. Part A : the journal of the International Society for Analytical 
      Cytology
JID - 101235694
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Biomarkers)
RN  - 0 (Cytokines)
RN  - 0 (Immunoglobulin Fab Fragments)
RN  - 0 (Receptors, IgG)
RN  - 0 (Toll-Like Receptor 2)
SB  - IM
MH  - Animals
MH  - Antibodies, Monoclonal/*immunology
MH  - Bacterial Infections/immunology
MH  - Biomarkers/metabolism
MH  - Cytokines/immunology
MH  - Flow Cytometry/instrumentation/*methods/standards
MH  - Humans
MH  - Immunoglobulin Fab Fragments/*immunology
MH  - Mice
MH  - Monocytes/cytology/*immunology
MH  - Receptors, IgG/immunology
MH  - Toll-Like Receptor 2/*immunology
EDAT- 2011/03/25 06:00
MHDA- 2011/07/02 06:00
CRDT- 2011/03/25 06:00
PHST- 2010/07/07 00:00 [received]
PHST- 2010/10/02 00:00 [revised]
PHST- 2010/10/29 00:00 [accepted]
PHST- 2011/03/25 06:00 [entrez]
PHST- 2011/03/25 06:00 [pubmed]
PHST- 2011/07/02 06:00 [medline]
AID - 10.1002/cyto.a.21000 [doi]
PST - ppublish
SO  - Cytometry A. 2011 Apr;79(4):247-55. doi: 10.1002/cyto.a.21000. Epub 2010 Dec 23.