A method for the immobilization of antibodies to inert matrix represents an important factor that affects results of immunoaffinity chromatography. Binding antibodies to immobilized metal ions is an example of oriented immobilization that avoids a random coupling of a protein. Preparation of a stable immunoaffinity sorbent using immobilized metal ions was described. Antibodies were bound to chelated Co3+ ions that were prepared by oxidation of Co2+-iminodiacetic acid agarose using hydrogen peroxide. The formation of a stable complex of the antibody with immobilized Co3+ ions was proved. Antibodies bound by this way were not released with solutions of 50 mM EDTA, 6 M urea, 3 M NaCl, 20% v/v dioxane, 0.1 M imidazole and buffers of pH 2.5 and pH 11.0. If needed, antibody could be released from the carrier by the reduction of Co3+ ions with a reducing agent (e.g. dithiotreitol or 2-mercaptoethanol). Antibody released from the carrier could be then replaced by another antibody. The method described in this paper was used for the immobilization of polyclonal rabbit anti-ovalbumin antibody or egg yolk antibody (IgY) produced in chicken. In a model experiment, immobilized polyclonal rabbit antibodies were used for the separation of ovalbumin from egg white and conditions of chromatography were described.

Institute of Pathophysiology 1st Faculty of Medicine Charles University Prague Czech Republic

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