Molecular Requirements for Polyamines Binding to the Antispermine Monoclonal Antibody Spm8-2

Abstract

The monoclonal antispermine antibody Spm8-2 was obtained by immunizing mice with a thyroglobulin-spermine conjugate. The molecular requirements for polyamines binding to this antibody were investigated by ELISA binding and inhibition tests, using a variety of natural polyamines and synthetic polyamine analogs. Four major structural determinants are important for the binding of polyamines by the antibody: (1) terminal amino groups: N-alkylation of both terminal amino groups of the polyamines leads to an important drop in the affinity for the antibody; (2) number of methylene groups spacing the amino groups: the four carbon chains appear to present the optimum length since the antibody binds polyamines with repeats of the aminobutyl moiety more actively than their homologues with shorter or longer carbon chains; (3) number of amino groups: the affinity of Spm8-2 for free homologous polyamines varied in the following order: pentamines > tetramines > triamines > diamines, showing the importance of the number of positive charges of the polyamines in the antibody-antigen reaction; the importance of charges is further emphasized by the dependence of antibody binding on the ionic strength of the medium; (4) N-acylation of one terminal amino group: the antibody binds more actively N1-acetylspermidine than spermidine or spermine. The binding properties of Spm8-2 suggest the presence of two recognition sequences, one selective for N-acylaminopropyl moieties, the second for the aminobutyl moiety.

Immunocytochemical demonstration of polyamines in nucleoli and nuclei

“…Hougaard et al (1987) have reported on nuclear localization of PAs in chick erythrocytes, and more recently, Johnson et al (2004) reported on nuclear staining for PAs in cell lines, although their data were not supported by speciWcity (absorption) controls. The latter authors used a weaker Wxation in 4% paraformaldehyde (PFA), and a mAb, which was produced against carbodiimide-conjugated Spm (Delcros et al 1996). These results are consonant with our data, which in addition, demonstrates that PAs are associated with nucleoli.…”

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

“…Due to the high amine residue content of TREN, the charge of the free amino groups could probably be very important in the binding of HSA and human IgG, following the order tetramines > diamines. The difference in K d values obtained for human IgG on TREN and on aminohexyl adsorbents is in accordance with studies developed by Delcros et al, which showed the importance of the charge (number of terminal amino groups) in the binding of polyamines to an antispermine monoclonal antibody [32].…”

Immunocytochemical localization of polyamines during attachment and spreading of retinal pigment epithelial and intestinal epithelial cells

“…Antibodies against polyamines were obtained from Dr. Jean Guy Delcros (Institute de Rechereche Contre le Cancer, Rennes, France). The antibody has been well characterized and was shown to have the highest binding affinity for spermine, with lesser affinity for spermidine and putrescine [Delcros et al, 1996;Bouille et al, 1999]. Antibodies against FAK were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.…”