Stripping of endogenous ligands from antisera against glucagon

In some antisera raised against glucagon in rabbits a large fraction of the antibody binding sites are occupied by glucagon-like material which appears to be of endogenous origin. Six rabbits were immunized with a glucagon-albumin conjugate, boosted twice and bled 5 times during the course of 72 days. The antisera were found to contain between 110-750 nmol/l antibody-bound glucagon. The concentration of bound glucagon as well as the concentration of free antibody binding sites (titre) increased with time in 5 rabbits. By gel chromatography the bound material co-eluted with intact pancreatic glucagon (as opposed to the immunogen). Five different methods were examined for their ability to remove the ligand. Charcoal treatment of the antiserum at pH 2.5 for 48 h removed 91 +/- 5% of the bound ligand. After stripping, binding capacities (titres) and binding affinity (equilibrium constants) amounted to 63.2 +/- 19.4% and 105.0 +/- 25.4% of the respective pre-stripping values.