FIG. 2.

(A) Cross-blocking of bhLF binding to solid-phase lipid A by uncoupled hLF. E. coli F 515 lipid A-coated wells were preincubated for 1 h with 100 μl of increasing concentrations (twofold increments) of uncoupled hLF (solid circles) or BSA (negative control; open circles) per well. Without removal of the competitor, bhLF (30 ng/well) was added, and binding was evaluated as reported in Materials and Methods. Nonspecific binding was subtracted. Data are representative results obtained in duplicate in three independent experiments. (B) Inhibition of bhLF binding to solid-phase lipid A by soluble lipid A. A constant amount of bhLF (final concentration, 300 ng/ml) was mixed with increasing concentrations (fourfold increments) of soluble E. coli F 515 lipid A (solid circles) or BSA (negative control; open circles) and incubated for 1 h. Then, 100 μl of this mixture was added to E. coli F 515 lipid A-coated wells, and binding was evaluated as reported in Materials and Methods. Nonspecific binding was subtracted. Data are representative results obtained in duplicate in three independent experiments.