Binding kinetics of soluble ligands to transmembrane proteins: Comparing an optical biosensor and dynamic flow cytometry

Background: The kinetics of protein-protein interactions can be monitored w ith optical biosensors based on the principles of either surface plasmon re sonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. Methods: We monitored with an IASys biosensor system, based on a resonant m irror: (1) the binding of cells to an immobilized ligand, (2) the binding o f a soluble ligand to immobilized cells, and (3) the binding of a soluble l igand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic f low cytometry. Results: With an optical biosensor, the useful configuration is the one bas ed on immobilized plasma membrane vesicles. However, signals can be detecte d only for very abundant binding sites (>10(6) per cell). Dynamic flow cyto metry allows the accurate determination of the k(on) and k(off) of antibody binding, The sensitivity of the method is two orders of magnitude better t han with an optical biosensor. Conclusions: Although biosensors constitute a method of choice for measurin g the interactions between soluble proteins, they are not well suited for m easuring the interaction between soluble proteins and membrane-embedded pro teins. On the contrary, flow cytometry is well suited for such an applicati on, when it is used in a dynamic mode. Cytometry 40:76-80, 2000. (C) 2000 W iley-Liss, Inc.