Molecular requirements for polyamines binding to the antispermine monoclonal antibody Spm8-2.
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.
Delcros
J G
JG
Laboratoire d'Immunochimie INSERM C.J.F.89-05, Faculté de Médecine Lyon Sud, Oullins, France.
Clément
S
S
Bouillé
N
N
Royou
A
A
Debroise
I
I
Thomas
V
V
Moulinoux
J P
JP
D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D011073:Polyamines / Q000378:metabolism*; D011700:Putrescine / Q000031:analogs & derivatives; D013095:Spermidine / Q000031:analogs & derivatives; D013096:Spermine / Q000031:analogs & derivatives / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011073:Polyamines; C106149:SPM8-2 monoclonal antibody; D013096:Spermine; D013095:Spermidine; D011700:Putrescine