Differential recognition of free and covalently bound polyamines by the monoclonal anti-spermine antibody SPM8-2

Abstract

The reactivity of an anti-spermine MAb (SPM8-2) toward polyamines either free or bound to a solid surface was investigated using equilibrium dialysis and ELISA methods. When polyamines were covalently linked to hydrophilized microtiter plates using carbodiimide, the MAb SPM8-2 reacted both with spermine and spermidine, with a higher affinity for the latter, but did not show any reactivity towards bound putrescine. In contrast, the MAb SPM8-2 reacted with all three polyamines bound to the microtiter plates with glutaraldehyde, with an affinity in the order: putrescine > spermidine > spermine. Equilibrium dialysis and competitive ELISA tests showed that the MAb SPM8-2 exhibited high affinity for free spermine and 50% and 5% cross-reactivity with free spermidine and putrescine respectively. The affinity of the MAb SPM8-2 for putrescine, spermidine and spermine appears to depend on whether the polyamine is free or bound. The antigenicity of the polyamines differs according to the nature of their link to the solid phase. These observations are discussed in the light of the structural modification produced by covalent binding of the polyamines. It is also concluded that when antibodies are used, due care has to be exercised in choosing the appropriate immunoassay for determining the specificity of antibodies directed against small haptens such as the polyamines.