Electrochemical investigation of hapten-antibody interactions by differential pulse polarography

The binding of electroactively labeled estriol with estrogen-specific antibody and its subsequent displacement by unlabeled estriol have been monitored by differential pulse polarography. Estriol was found to be electro-inactive in the potential range -200 mV to -1000 mV vs a silver/silver chloride electrode. Estriol labeled in the 2 and 4 position with nitro groups was electroactive, giving two reduction waves at -422 mV and -481 mV vs a silver/silver chloride electrode. The peak current was linear with concentration over the range 60 micrograms/L to 3.7 mg/L. The addition of aliquots of estrogen-specific antibody reduced the peak current proportionately, indicating the binding of ligand to specific antibody. Subsequent addition of unlabeled estriol produced incremental increases in peak reduction current, indicating competitive and reversible binding of the two ligands for the antibody. Separation of bound from free labeled hapten was not necessary because reduction of the antibody-bound labeled estriol is attenuated at the electrode.