No abstract

ed by subtracting the nonspecific binding from the total binding. To determine the degree of ligand depletion, aliquots were withdrawn from the supernatant in each well and counted by liquid scintillation. The experimental setup is depicted in Figure 1. hHBP binds 3 H-LPS in a saturable manner (Figure 2). By using scientific software, curvefits to the data were performed. The apparent K d was determined to be 90 ± 5 ng/mL and the B max to 0.2 ng 3 H-LPS/ng hHBP. The nonspecific binding increases by adding to the 3 H-LPS concentration in a direct proportional manner, as expected. In the absence of either hHBP or anti-hHBP antiserum, only negligible signals are produced (results not shown), which verifies that the observed signal is highly dependent on both hHBP and anti-hHBP antiserum. The ligand depletion was 40% at 25 ng/mL and 10% at 912 ng/mL. In our laboratory, this assay has been used for competitive studies. The experiments showed that LPS from different species were able to compete with 3 H-LPS binding to hHBP (results not shown). The results presented demonstrate that the assay is a powerful method for measuring LPS affinity of different proteins. Furthermore, whole cells or fragments can be incorporated instead of the purified hHBP. Most interestingly, the assay can be used in screening programs for enhancers and inhibitors of LPS binding.