A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model

Abstract

Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-L-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N1-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N1-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.