Multiple epitope specificity of monoclonal antibodies to a single synthetic peptide: use in the characterization of the GP IIb-IIIa binding domain of von Willebrand factor.
Monoclonal antibodies have been induced against the synthetic peptide with sequence Tyr-Glu-Val-Val-Thr-Gly-Ser-Pro-Arg-Gly-Asp-Ser-Gln-Ser-Ser. This peptide represents residues Glu1737-Ser1750 of the mature von Willebrand factor (vWF) subunit and contains the sequence Arg-Gly-Asp, thought to be important in mediating binding to the platelet receptor glycoprotein (GP) IIb-IIIa complex. Twelve antibodies were obtained, eight of which bound to native vWF as well as to the peptide immunogen insolubilized onto agarose beads. These antibodies defined at least three distinct epitopes, as demonstrated by antibody interaction with peptides having a single phenylalanine substitution at each position in the sequence. In particular, two antibodies bound to epitopes on vWF that included one or more of the three residues (arginine, glycine, aspartic acid) thought to be involved in binding to GP IIb-IIIa, whereas one antibody bound to an epitope that did not include any of those residues. Nevertheless, the three antibodies cross-reacted with each other, a finding explained by the fact that the corresponding epitopes had at least two residues in common, namely Gly1741 and Ser1742. In spite of the cross-reactivity for binding to vWF, only the two antibodies whose epitopes included residues in the Arg-Gly-Asp sequence inhibited vWF interaction with GP IIb-IIIa. The third antibody had no inhibitory effect even though it was bound to an epitope located at a distance of only few residues on the amino terminal side of Arg-Gly-Asp. These results demonstrate that monoclonal antibodies raised against a single synthetic peptide with sequence limited to fifteen residues may exhibit distinct epitope specificity and may be used to define functional domains in macromolecules with a high degree of resolution.
Berliner
S A
SA
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Houghten
R A
RA
Roberts
J R
JR
Ruggeri
Z M
ZM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis*; D010973:Platelet Adhesiveness; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor