Antibody-metal ion conjugates

Deferoxamine
DFA or deferoxamine is a naturally occurring product is isolated from Streptomyces pilosus. Its native activity is forming iron complexes The amine group of DFA can be utilized for direct labeling of antibodies and other proteins through coupling with available carboxylates using carbodiimide-mediated conjugation with EDC

 It has medical applications as a chelating agent used to remove excess iron from the body. The mesylate salt of DFO-B is commercially available It can also bind to aluminium or other trivalent atoms.

Carboiimide mediated conjugation

Carbodiimides are used to mediate the formation of amide linkages between a carboxylate and an amine or phosphoramidate linkages between a phosphate and an amine.

EDC
EDC is perhaps the most popular carbodiimide for use in conjugating biological substances. Its water solubility allows for direct addition to a reaction without prior organic solvent dissolution.

Procedure
Dissolve the protein to be modified at a concentration of
10 mg/ml in one of the following reaction media: (a) water, (b) 0.1 M MES, pH 4.7-6.0, or (c) 0.1 M sodium phosphate, pH 7.3. NaCl may be added (i.e., 0.15 M) if desired. If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants added as necessary to maintain the correct molar ratios. For the preparation of a peptideprotein immunogen conjugate, a 200-uL solution of the carrier protein at a concentration of 10 mg/ml in 0.1 M MES, pH 4.7, usually works well.

Dissolve the molecule to be coupled in the same buffer used in step 1. For small molecules, add them to the reaction in at least a 10-molar excess to the amount of protein present. Add EDC (Pierce) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.5-0.1 M EDC concentration in the reaction usually works well. React for 2 hrs at room temperature. Use gel permeation chromatography or dialysis to purify the conjugate from isourea formed.

Alternative reagents
EDC plus Sulfo-NHS CMC Diisopropyl carbodiimide Dicyclohexyl carbodiimide (DCC)

Woodward's reagent N'-N'-Carbonyldiimidazole

Use of homobifunctional cross linkers

The first cross-linking reagents used for modification and conjugation of macromolecules consisted of bireactive compounds containing the same functional group at both ends. Like molecular rope, these reagents could tie one protein to another by covalently reacting with the same common groups on both molecules. The main diadvantage of using simple homobifunctional reagents is the potential for creating a broad range of poorly defined conjugates.

Advantage of homobifunctional cross-linkers

Can cross link most type of molecules as hundreds of different compounds have been developed. Do not affect immunospecificity or immunoreactivity.