The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated.