Oriented covalent immobilization of antibodies onto heterofunctional agarose supports: A highly efficient immuno-affinity chromatography platform

The development of new bioconjugates formed by one antibody optimally bound (through its Fc region) to fairly inert solid surfaces is of primary relevance in immuno-affinity chromatography. Immunoglobulins G (IgG) have a Fc region very rich in histidine (His) residues. In this way, immobilization of IgGs on heterofunctional metal chelate-glyoxyl supports (Ag–Me2+/G) takes place in two steps: firstly the antibodies are conjugated to the support via His–metal coordination bonds. Secondly, their incubation under alkaline condition promotes an intramolecular covalent attachment between lysine residues at the Fc region and glyoxyl groups on the support surface. The IgG that recognizes as antigen the HRP (antiHRP–IgG) has been conjugated to Ag–Me2+/G supports. The resulting bioconjugate is highly inert and able to specifically bind the antigen (HRP) without significant unspecific binding of any other proteins, resulting in an excellent HRP purification platform. The binding activity of this bioconjugate has been optimized by controlling the antibody distribution throughout the bead's surface in order to avoid high antibody densities that led to a low binding activity of the antibodies. The optimal antibody distribution has been achieved when these proteins were slowly immobilized on Ag–Cu2+/G in presence of imidazole. This bioconjugate was able to bind up to 1.5 moles of antigen per mole of antibody, only 1.3-fold less than the antibody in solution. Hence, we have been able to develop an optimal protocol to prepare bioconjugated composites in an oriented and irreversible fashion which results in highly efficient and specific surfaces for the exclusive biological recognition.

► Oriented and irreversible conjugation of antiHRP–IgG to agarose beads.
► Agarose beads heterofunctionally activated with both glyoxyl and metal chelate groups.
► Inert bioconjugates with low unspecific binding of any other proteins.
► Bioconjugates with low antibody density present higher biological activities.
► Antibody density and distribution was controlled by the antibody immobilization rate.