Premium
Immunoaffinity carrier prepared by immobilization of antibody via Co 3+ ‐chelate
Author(s) -
Zatloukalová Eva,
Kucerová Zdenka
Publication year - 2006
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500466
Subject(s) - chemistry , iminodiacetic acid , affinity chromatography , polyclonal antibodies , chelation , chromatography , hydrogen peroxide , agarose , metal ions in aqueous solution , ovalbumin , sorbent , nuclear chemistry , antibody , metal , inorganic chemistry , adsorption , biochemistry , organic chemistry , antigen , immunology , biology , enzyme , genetics
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 Co 3+ ions that were prepared by oxidation of Co 2+ ‐iminodiacetic acid agarose using hydrogen peroxide. The formation of a stable complex of the antibody with immobilized Co 3+ 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 Co 3+ 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.