An evaluation of polyamine immunocytochemistry using immunocytochemical model systems

Polyamine (PA) immunocytochemistry (ICC) was evaluated by a recently developed enzyme-linked immunosorbent assay (ELISA) binding test (ELISABT) using an anti-spermine (Spm) serum raised against Spm conjugated via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide (GMBS) with bovine serum albumin. In the test the antiserum showed strong immunoreactivity with N1-acetylspermine (Ac-Spm) and acetylspermidine (N1-Ac-Spd and N8-Ac-Spd), and low immunoreactivity with Spm and Spd, which was, however, markedly enhanced after reaction with GMBS, acetic anhydride or glutaraldehyde. Complete agreement with results of immunoblot analysis was observed. PA-like immunoreactivity observed in the present PA ICC in cells in the foveolae and isthmus of rat gastric glands was completely abolished by absorption of the serum with N1,N12-diacetyl-Spm, Ac-Spm or N1-Ac-Spd, but not by Spm or Spd. This absorption test was then simulated by an ELISA inhibition test (ELISAIT) with a solid phase conjugated with Ac-Spm or Spm, and by a PA ICC model system using Sepharose gel beads conjugated with each of several PAs. The results strongly suggest that the immunostaining in the gastric mucosa was mainly due to antibody species in the serum specific to 'acylated' Spm and Spd, but not to Spm or Spd. Acetyl PAs exist at such low concentrations in animal tissues that they are virtually undetectable by current ICC methods. Therefore Spm and Spd are likely candidates for those detected, after having been converted by fixation into such PA derivatives as become reactive with the antiserum.