Determination of the association constant and the maximum binding capacity of radionuclide labelled monoclonal antibody

Objective To determine the maximum number of McAb molecules bound by per antigen cell and the association constant of the binding reaction. Methods Using binding assay for binding of radionuclide labelled McAb with target cell antigen, the formula of equilibrium plot was T/B=1+1/ K a·C+T/C (1) and F/B=1/ K a·C+F/C (2), K a,T,B,F and C were the association constants, the concentration of total, binding, free McAb and constant cell antigen volumn, respectively. The experimental data demonstrated a very significant linear correlationship of formula(1) T/B to T and formula (2) F/B to F and that was calculated by means of linear regression analysis. Results The inverse T/B value of intersecting point of formula (1) straight line with the T/B axis was the immunoreactive fraction of labelled McAb. Using constant labelled McAb concentration and different unlabelled McAb substitute for T, the distance from the intersecting point of this line with T/B value of immunoreactive fraction to T/B axis was the actual concentration of immunoreactive fraction of labelled McAb. The maximum number of McAb molecules bound by per antigen cell was the inverse of the slope of formula (2)straight line and the association constant was the slope divided by intercept of formula (2) straight line with F/B axis. Conclusion The method is feasible for determination of the association constant and the maximum number of McAb molecules bound by per antigen cell.