In the case of the Le x glycocalyx model binding to CTB, there was a larger 11,000-fold enhancement in binding relative to the 10 mM Kd reported in the literature for the monovalent interaction. In this case, the binding sites are arranged around the periphery of the protein which should be better disposed to multivalent binding to the 3D glycocalyx model. Here, the glycan:CTB pentamer ratio was approximately 30 at saturation, even though it is not feasible for a CTB pentamer to engage with 30 copies of Le x . Our quantitative analysis (Table , Figure and Figure ) shows that the combined mass concentration of glycopolymers and proteins in the model glycocalyx does not exceed 130 mg/mL, implying that solvent typically constitutes more than 90% of the film volume and that the film is spacious enough to accommodate more protein. We therefore conclude that the lack of Le x glycan saturation in the model glycocalyx could arise from steric occlusion of glycans that are too close to the first CTB pentamer to allow their interaction with additional pentamers, and/or from unfavourable entropic effects of sterically constraining the glycopolymers in the glycocalyx upon binding (i.e., reduced conformational entropy).