Ferrocene is representative of an unique class of compounds, the metallocenes, which incorporate a heavy metal atom in a pseudo-aromatic cyclopentadienyl ring system of benzene-like stabillity. Two ferrocenyl groups per polypeptide chain slightly enhance the amount of antibody elicited, but only a small amount of the antibody is directed at the ferrocenyl group. At this level, ferrocene is useful as a label for metabolic studies. Larger amounts of ferrocene (10 to 16) show strong hapten activity with up to 70% of the antibody directed at the ferrocenyl group. The ferrocene-antiferrocene reaction can be inhibited by ferrocenyl-acetic acid. In addition, the heavily labelled molecules elicit larger amounts of antibody than the parent polymer or the derivative containing 2 ferrocenes.

The ability of ferrocene to enhance immunogenicity reflects behavior similar to that of the aromatic amino acids and cyclohexylalanine. Ferrocene may be even more unique in this regard, because it enhances immunogenicity in a polypeptide which contains 6% tyrosine and is already a good immunogen.

The metallocenes, of which ferrocene is a prototype, have three distinct advantages for use in metabolic studies: they incorporate radioactive heavy metal atoms with relatively long half-lives; they are biologically stable and probably cannot be reutilized by the animal in any significant amount; and they may become electron-dense if short polymers of the metallocenes are attached to the polypeptide chain.

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