Figure 1.

(a) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. (b) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.