Immunoelectron microscopy study of polyamines using a newly prepared monoclonal antibody against spermidine: use of a mixture of glutaraldehyde and paraformaldehyde as a cross-linking agent in the preparation of the antigen

Abstract

We developed a mouse monoclonal antibody (ASPD-19, IgG3 sub-isotype mAb) against spermidine (Spd) conjugated to bovine serum albumin (BSA) using a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA)-sodium borohydride for applications in immunoelectron microscopic studies. The antibody specificity was evaluated by an ELISA binding test simulating the immunocytochemistry (ICC) of tissue sections. The ASPD-19 mAb is highly specific for Spd and Spm, almost the same degree to each, and can distinguish alterations in the chemical structure of other polyamine (PA) analogs, showing less than 3.2% cross-reaction with N(1)-acetylspermidine, acetylspermine, or N(8)-acetylspermidine. By an indirect immunoperoxidase method using the ASPD-19 mAb, PA-like immunoreactivities were observed in different tissues fixed with Karnovsky fixative (a mixture of GA and PFA) in combination with borohydride reduction. In contrast, immunoreactivity was very low in tissues when the borohydride reduction step was omitted. The PA-like immunoreaction was completely abolished by the adsorption of the ASPD-19 mAb with 100 microg/ml of Spd or Spm, but was inhibited little or none by other PA-related compounds or amino acids. A light microscopic ICC method using ASPD-19 produced immunostaining of PAs in certain cells in rat tissues with high biosynthetic activities (small intestine, pancreas and spinal cord). A pre-embedding immunoelectron microscopic study using rat spinal cord showed PA immunoreactivity located predominantly on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm of motor neurons. These results are in complete agreement with the results obtained by our recent ICC method using another mAb (ASPM-29) produced against GA-conjugated Spm.