RECOVERY OF MONOCLONAL-ANTIBODY FROM ITS COMPLEX WITH LIGAND

The separation of antibody from its excess and bound ligand is importa nt following affinity chromatography or the use of methods requiring l arge amounts of antibody, such as microcalorimetry. Using radioactivel y labeled ligands we show that these separations can be effected by us ing commercially available, short polyacrylamide size-exclusion column s. By using both low (k(a) = 5 x 10(2) M(-1)) and medium high-affinity (K-a = 0.6 x 10(6) M(-1)) ligands in the presence of antibody, it is shown that the latter system requires more dilute loading concentratio ns than the former system does in order to achieve acceptable separati on. Since the degree of association between a protein and a ligand is solely governed by the affinity constant for the binding equilibrium, these results are applicable to any system represented by this range o f binding constants.