Site-directed immobilization of proteins.

To determine if immobilization chemistry can be used to orient antibody on a support so that the bivalent binding potential can be fully utilized, we developed three activated matrices that couple to different functional groups on the molecule. When AminoLink Gel was used to couple antibody randomly through primary amino groups, the molar ratio of immobilized antibody to recovered antigen averaged 1:1. Iodoacetyl groups on SulfoLink Gel couple through sulfhydryls in the hinge region of the antibody molecule, in theory leaving the antigen binding site available. However, the antibody-to-antigen molar ratio was only slightly improved. Hydrazide groups on CarboLink Gel couple to aldehyde groups generated by oxidation of carbohydrate moieties that are located primarily on the Fc portion of the antibody molecule. The molar ratio of immobilized antibody to purified antigen using CarboLink Gel reached the optimum of 1:2. CarboLink Gel is most effective at orienting antibody for better antigen purification capability.