Complexes between monoclonal antibodies and receptor fragments with a common cold virus: Determination of stoichiometry by capillary electrophoresis

Complex formation between monoclonal antibodies or soluble receptor fragmen ts and a human rhinovirus is quantified by relating the concentration of th e antibody or receptor under equilibrium conditions to the initial concentr ation of the virus. Within a given concentration range of the reactants, th e shape of the resulting curve depends only on the value of the dissociatio n constant of the particular system studied. Using antibodies and receptor fragments, cases for high, low, and intermediate affinity were investigated . For high-affinity systems, the curve approximates a decaying straight lin e and the binding stoichiometry can be accurately determined from the inter cept with the x-axis. For the case of intermediate affinity, the curve can be linearized at low virus concentrations with the receptors present in lar ge excess. Extrapolation of this line allows derivation of the binding stoi chiometry from the intercept with the x-axis, although with less accuracy. For intermediate affinities, an estimate of the dissociation constant can b e obtained from fitting the curve to the data points measured. Finally, in the case of low affinity none of the binding parameters can be quantified, although a rough estimate of the lower limit of the dissociation constant i s possible. The method was applied for two different monoclonal antibodies, a Fab fragment and a receptor fragment, binding to human rhinovirus seroty pe 2. Thirty copies of the monoclonal antibody 8F5 were found to bind to th e virion, which is in agreement with data from electron cryomicroscopy. The complex between monovalent human very-low-density lipoprotein receptor enc ompassing repeats 2 and 3 and human rhinovirus serotype 2 showed 60 recepto r molecules bound per virion.