Binding of MD-2 to the surface of Yp is independent of serum components. (A) The indicated amounts of live bacteria (x-axis) were incubated in HBSS with a fixed amount of MD-2^6xHis (input). MD-2 in the postcellular supernatants (y-axis) was quantitated by ELISA (B) and plotted as a function of the number of bacteria used in the binding reaction (x-axis). About 10⁷ bacteria were necessary to deplete 20 ng of MD-2^6xHis input, which corresponds to about 40 000 MD-2 binding sites on the surface of Yp, assuming that bacteria have 10⁶ molecules of LPS on their surfaces. Plotted values are the average of 2 independent experiments plus or minus the range. (B) Soluble MD-2 form the serum of healthy donors (n = 10) was determined by ELISA using plastic adsorbed TLR4-Fc to capture sMD-2 and a commercial α–MD-2 mAb as detection reagent. (C) Binding isothermes were generated as in panel A, but here human serum was used as a source of sMD-2. Plotted are sera from 3 representative donors. Error bars are the SD of triplicate determinations.