Binding parameters of antibodies - a reappraisal

Reliable methods of determining antibody avidities are essential for compar ing different antibodies (Abs) and for evaluating novel Ab constructs. Alth ough it is accepted that true affinity applies only to monovalent binding, the concept of 'functional affinity' has been widely applied to multivalent binding interactions. We herein summarize the data indicating that 'functi onal affinities' are invalid. In many cases, binding of IgG Abs to multival ent antigens such as the cell surface is essentially irreversible, which is a consequence of bivalent binding and which implies that use of 'functiona l affinity', which means 'functional equilibrium association constant', can not be valid. Alternative methods of evaluating Ab avidity are proposed. Wi dely used methods to measure either the total number of binding sites per c ell or the immunoreactive fraction of Ab are also unreliable, primarily bec ause of their dependence on extrapolation.