A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4

Figure 3

M-860 recognizes a conformational epitope of huLTF. (a) Representative histograms showing the staining of FITC-labeled M-860/L3262 on gated T lymphocytes or huPMNs. After lysis of erythrocytes, total leukocytes in human peripheral blood were stained with FITC-labeled M-860/L3262 before FACS analysis. FITC-labeled mIgG1/RbIgG were included as negative controls. Lymphocytes and huPMNs were gated according to their distinct forward- and side-scatter properties. (b) M-860 does not recognize huLTF in western blot analysis. Total cell lysates of huPMNs (2 μg, Lane 1), recombinant human (50 ng, Lane 2), murine (200 ng, Lane 3), or bovine (200 ng, Lane 4) LTF were subjected to SDS-PAGE in the presence of 2-ME. After the transfer to a PVDF membrane, the membrane was blotted with M-860 or L3262 overnight at 4°C, followed by incubation with proper HRP-coupled secondary antibodies. (c) M-860 does not recognize denatured huLTF in ELISA. Serially diluted M-860/L3262 or corresponding control Abs (indicated in -axis) were incubated in wells precoated with 2-ME-treated (1% 2-ME for 2 hrs, upper panel) or heat-treated (95°C for 10 mins, lower panel) huLTF, followed by routine ELISA procedures. (d) M-860 pulls down huLTF from total huPMNs lysates. Total cell lysates of huPMNs were incubated overnight with 1 μg mIgG1 (Lane 1), M-860 (Lane 2), RbIgG (Lane 3), or L3262 (Lane 4) at 4°C, followed by incubation with protein G-sepharose. The precipitated proteins were subjected to western blot analysis with L3262 as the detection Ab. Recombinant huLTF (50 ng, Lane 5) was included as a positive control.