A new graphical method for determining the affinity constants of monoclonal antibodies to enzymes

A new graphical method is presented for determining the affinity constants of antibodies to enzymes. The method does not require purification of reactants or separation steps at equilibrium. The plot 1/v versus [AT]/V - v) ([AT] = total concentration of antibody binding sites, V = enzyme activity measured in the absence, and v = activity measured in the presence, of antibodies) yields straight lines in the case of simple antibody-enzyme interactions. More complex interaction models show different curve shapes, which can be used for model discrimination as shown by computer simulation studies. The applicability of the method was demonstrated by the determination of the affinity constant of the monoclonal antibody IB 10B8 to alkaline phosphatase of calf intestine. This antibody inhibits enzymic activity completely.