Proton-n.m.r. study of interaction of myelin basic protein with a monoclonal antibody.
Proton n.m.r. at 400 MHz has been applied to study the interactions of bovine or porcine myelin basic protein (b- or p-MBP) with a monoclonal antibody to human (h-) MBP. The antibody, an IgG immunoglobulin that contains a sequential epitopic region, cross-reacts with b-MBP but not with p-MBP, the presumed epitope being identical in h- and b-MBP. N.m.r. spectra were recorded from the Fab fragment of the antibody and for mixtures of Fab and MBP at various molar ratios. The n.m.r. spectrum of MBP in the mixture consists mostly of well resolved peaks against a broad background due to the Fab. With b-MBP, but not p-MBP, specific interactions are observed at the residue tyrosine-135, which is part of the epitopic sequence. Other interactions occur between the Fab and both b- and p-MBP at residues distant from the epitopic region. Standard radioassay techniques were employed to calculate the binding constants of both basic proteins with the immunoglobulin. The binding constant, Kb, for IgG to column-immobilized b-MBP at 298K is (0.95 +/- 0.07) X 10(7) dm3/mol. The value of Kb decreases with the ionic strength of the medium, suggesting a coulombic interaction between antigen and antibody. N.m.r. spectra were also measured for mixtures of the Fab fragment and peptides containing the epitopic site, with results in agreement with those for the whole protein.
G L
GL
Mendz
W J
WJ
Moore
S
S
Easterbrook-Smith
D S
DS
Linthicum
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002942:Circular Dichroism; D003429:Cross Reactions; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D004676:Myelin Basic Protein / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D004676:Myelin Basic Protein; D010446:Peptide Fragments