Experimental Study of the Rate of Bond Formation Betwwen Individual Receptor-Coated Spheres and Ligand-Bearing Surfaces

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Abstract
The efficiency of cell adhesion is highly dependent on the rate of association between adhesion molecules when membranes are at bonding distances. Whereas kinetic parameters of interactions involving at least one soluble molecular species have been extensively studied, the definition and experimental determination of corresponding parameters when both receptors and ligands are bound to surfaces are much more difficult to achieve. In the present work, we explore the feasibility of measuring the rate of association between antibody-coated spheres and antigen-derivatized surfaces in presence of an hydrodynamic shear force lower than the strength of a single bond. An image analysis procedure allows continuous recording of particle position with about 0.05 μm accuracy and a time resolution of 5 milliseconds. We present an original procedure allowing direct determination of the wall shear rate by processing the images of moving spheres. Further, simultaneous determination of the Brownian fluctuations perpendicular to the bulk fluid motion and the mean translational velocity of particles allows in principle a numerical determination of the sphere-to-substrate distance within a range of about 10 to 1000 nm. It is concluded that: i) particle motion is in rough agreement with current hydrodynamic theories based on creeping flow approximation. ii) In our experimental system adhesion seems to be diffusion-limited, therefore, only a lower boundary for the kinetic constant of molecular association can be obtained. iii) Further improvement of our method will require the production of molecularly smooth receptor-coated surfaces

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