Metal complex-antibody conjugates

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

The linking of a metal to an antibody could, in principle, be accomplished by forming the metal chelate either prior to or after attachment to protein. Success to date has been achieved only by formation of the protein-ligand conjugate before metal chelation. The complexation reaction has several general features. First, reactions between the metallic radionuclides and antibodies are almost always performed with sub-stoichiometric quantities of chelate and metal ion. It is therefore of the utmost importance that no carrier added metals obtained from commercial sources be exceedingly pure or else be purified prior to use. Reactions of "carrier added" metal solutions are not likely to be of use because of the ease with which available chelate sites become saturated. Because the formation of chelate complexes is usually a bimolecular reaction, the complexation will proceed optimally when more chelation sites are available. Similarly, the more isotope in solution, the faster the reaction. Employment of a carrier chelate to insure solubilization of the radiometal is of value to maximize available isotope and the acetate ion has proven useful. 

There are two methods the preformed radiometal-chelate method and the indirect chelator—antibody method. Various antibodies are labelled by the latter, where the bifunctional chelating agent is initially conjugated to a macromolecule, which is then allowed to react with a metal ion, to form a metal-chelate-macromolecule complex. Due to the presence of the chelating agent, the biological properties of the labeled protein may be altered and must be assessed before clinical use. 

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