Irreversible, oriented immobilization of antibodies to cobalt-iminodiacetate resin for use as immunoaffinity media

A method for the construction of antibody affinity columns has been developed which utilizes the innate affinity of antibodies for metal. Antibodies that are reversibly bound to a cobalt-iminodiacetate resin are attached in an "exchange inert" fashion when the cobalt is oxidized from the 2+ to 3+ state. Antibodies bound in this fashion are not removed by metal chelating reagents, high salt, detergents, or chaotropic agents. Only reagents which reduce the metal are capable of removing the antibody. Since the metal binding site in antibodies is in the C-terminus of the heavy chain, antibodies bound to the resin are oriented with the combining site directed away from the resin thus allowing maximal antigen binding. The oxidation conditions used are mild and do not subject the antibody to harsh immobilization chemistry. This technique may be used to construct affinity media using multiple subclasses of antibodies since the metal binding site is conserved. This immobilization strategy may be a general technique for the oriented immobilization of antibody to surfaces.