Human serum–derived MD-2 binds to bacteria. (A) PAS-conjugated TLR4-Fc or live Yp cells were used to precipitate MD-2 from the serum of a healthy individual or 15 mL of baculoviral supernatants containing MD-2^6xHis as a positive control. The pellets were washed and Western blotted for the presence of MD-2 using a commercial α–MD-2 mAb, followed by an HRP-conjugated α-mouse antiserum and ECL. The 160-kDa band in the uppermost panel is TLR4-Fc, which is recognized by the α-mouse antiserum. Similar results were obtained with 2 other healthy donors. Vertical lines have been inserted to indicate repositioned gel lanes. (B) Live Yp cells were stained with PHS (left panels), PHS that was depleted of MD-2 using TLR4-Fc (middle panels), and depleted PHS that was reconstituted with recombinant MD-2 (20 ng/mL; right panels). Bound MD-2 was detected by FACS using either TLR4-Fc (top panels) or an α–MD-2 mAb followed by an FITC-labeled α-mouse pAb. The shaded profiles represent the binding of the secondary reagent without MD-2 coating. Fluorescence intensity is plotted in a log scale (x-axis; 10-10⁵). (C) Bacteria were adhered to plastic in 2-fold dilution and incubated with PHS. Human serum–derived soluble MD-2 bound to the surface of the bacteria was revealed by ELISA as in Figure 1C. Data are averages of triplicates plus SD.