Affinity of nonhomologous amphiphilic peptides toward a monoclonal antibody raised against apolipoprotein A-I.
Monoclonal antibodies against human apolipoprotein A-I (apoA-I) were generated by the hybridoma technique. Clone G-10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme-linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA-I bound to the surface of the microtiter plate, apoA-I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA-I/anti-apoA-I-complex, Kd = 2 x 10(-9) M, was significantly lower than that of the complex resulting from the interaction between anti-apoA-I and either apoA-I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G-10 recognizes in antigenic peptides an alpha-helical secondary structure of defined hydrophilic-lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010455:Peptides / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D009842:Oligopeptides; D010455:Peptides