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

Figure 1

The purity, subtype, and specificity/affinity of M-860. Seven to twelve days after the implantation of hybridoma cells, ascites from BALB/c mice were collected and immunoglobulin G was purified by protein G-sepharose affinity columns. The purified IgG, M-860, was dialysed against PBS overnight at 4°C before the analysis for purity, subtype, and specificity/affinity by SDS-PAGE (a) or LTF-based ELISAs (b and c). (a) SDS-PAGE showing the purity of isolated M-860 antibody. Lane M: marker; Lane 1: purified M-860. (b) M-860 belongs to mouse IgG1 subtype and binds to huLTF in ELISA. M-860 (1 μg/mL) or total mouse IgG (Ctrl) was incubated in ELISA wells precoated with huLTF for 2 hrs at 37°C. After washing, HRP-coupled goat Abs recognizing mouse IgG1, IgG2a, IgG2b, or IgG3 (indicated in -axis) were added and incubated for 1 hr. Signals were visualized at OD 492 nm by an ELISA reader after the addition of OPD. (c) Line graphs showing the specificity and affinity of M-860 for huLTF. Different concentrations (as indicated in -axis) of M-860, L3262, and their corresponding control Abs were incubated in ELISA wells precoated with huLTF (left panel) or bovine LTF (boLTF, right panel). Signals were visualized at OD 492 nm by an ELISA reader after sequential incubation with HRP-coupled goat anti-mouse/rabbit total IgG and then with OPD.

(a)

(b)

(c)