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 fragments and a human rhinovirus is quantified by relating the concentration of the antibody or receptor under equilibrium conditions to the initial concentration of the virus. Within a given concentration range of the reactants, the shape of the resulting curve depends only on the value of the dissociation 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 line and the binding stoichiometry can be accurately determined from the intercept with the x-axis. For the case of intermediate affinity, the curve can be linearized at low virus concentrations with the receptors present in large excess. Extrapolation of this line allows derivation of the binding stoichiometry from the intercept with the x-axis, although with less accuracy. For intermediate affinities, an estimate of the dissociation constant can be 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 is possible. The method was applied for two different monoclonal antibodies, a Fab fragment and a receptor fragment, binding to human rhinovirus serotype 2. Thirty copies of the monoclonal antibody 8F5 were found to bind to the virion, which is in agreement with data from electron cryomicroscopy. The complex between monovalent human very-low-density lipoprotein receptor encompassing repeats 2 and 3 and human rhinovirus serotype 2 showed 60 receptor molecules bound per virion.
Okun
V M
VM
Institute of Analytical Chemistry, University of Vienna, Austria.
Moser
R
R
Blaas
D
D
Kenndler
E
E
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology*; D012229:Rhinovirus / Q000737:chemistry / Q000276:immunology*; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments