Binding properties of monoclonal anti-IgG antibodies: analysis of binding curves in monoclonal antibody systems.

The binding properties of an immune complex-forming system comprising human IgG and mouse monoclonal antibodies against human IgG have been studied. A refined binding assay has been applied directly on ascitic fluid containing monoclonal antibody. Complete sets of binding data of a series of different monoclonal antibodies were collected and analysed by various graphical and statistical methods. Special attention was given to methods which allow determination of specific monoclonal antibody concentration as well as antibody affinity. It was found that the formation of genuine antigen: antibody complexes per se gives rise to deviations from expected linearity in commonly used binding equations. Good correlation was found between the antibody concentrations obtained by various graphical approaches, whereas the size of the association constant seemed to depend on the method in use. The binding pattern was found to be dependent on the concentration of antibody. Most reliable parameters were obtained if the product of the antibody concentration and the association constant was below 10.