K. Fujiwara et al., A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry, HISTOCHEM C, 115(6), 2001, pp. 471-477
We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb
) against putrescine (Put) conjugated to bovine serum albumin using a gluta
raldehyde (GA)-sodium borohydride procedure, for applications in immunocyto
chemistry (ICC). The antibody specificity was evaluated by an ELISA binding
test, simulating the ICC of tissue sections. APUT-32 mAb was highly specif
ic to Put, and distinguished alterations in the chemical structure of other
polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% w
ith spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results
in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition
test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-li
ke immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell
lines fixed with GA in combination with NaBH4 reduction, but almost no imm
unoreaction was seen in the cytoplasm of the human melanoma ED cell line. O
n the other hand, the same method but using a previously prepared ASPM-29 m
Ab, specific for spermine (Spm) and Spd, produced intense immunostaining in
the cytoplasm of all the three cell types. The Put-like immunoreaction was
completely abolished by absorption of the APUT-32 mAb with 10 mug/ml Put-h
uman serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was
also performed for the levels of each of the PAs in the three types of cel
l, showing that the levels of Put detected were much lower than those of Sp
m and Spd, and were strikingly different in the three cell lines among whic
h the human melanoma ED cell line contained the lowest levels of Put. These
results strongly suggest that APUT-32 mAb reacts specifically with Put in
the tumor cells and therefore has the potential as a new tool for elucidati
ng the biological roles of Put in cells and tissues.