We used a combination of laminar flow chamber and reflection interference microscopy to study the formation and rupture of single bonds formed between Fc-ICAM-1 attached to a substrate and anti-ICAM-1 carried by micrometric beads in the presence of a repulsive hyaluronan (HA) layer adsorbed onto the substrate. The absolute distance between the colloids and the surface was measured under flow with an accuracy of a few nanometers. We could verify the long-term prediction of classical lubrication theory for the movement of a sphere near a wall in a shear flow. The HA polymer layer exerted long-range repulsive steric force on the beads and the hydrodynamics at the boundary remained more or less unchanged. By incubating HA at various concentrations, the thickness of the layer, as estimated by beads most probable height, was tuned in the range 20-200 nm. Frequency of bond formation was decreased by more than one order of magnitude by increasing the thickness of the repulsive layer, while the lifetime of individual bonds was not affected. This study opens the way for further quantitative studies of the effect of molecular environment and separation distance on ligand-receptor association and dissociation.