Preparation of monoclonal antibodies to hirudin and hirudin peptides. A method for studying the hirudin--thrombin interaction.
A panel of four monoclonal antibodies was obtained against hirudin, a potent and specific inhibitor of thrombin, by immunizing three groups of mice with protein conjugates made of recombinant desulfatohirudin (group I) or two synthetic peptides representing the C-terminal sequences 40-65 (group II) and 52-65 (group III) of hirudin. Only the monoclonal antibody 4049-83-12, obtained from the group I of mice, showed high affinity for hirudin (Kd of 0.6 nM) and in vitro neutralizing properties. The anti-peptide monoclonal antibodies bound hirudin with lower affinity (Kd of 1.5-7 nM) and showed lower neutralizing capacities. An epitope analysis performed by competitive ELISA using various hirudin analogues and by limited proteolysis of the hirudin-antibody complex revealed that the binding domains of all the anti-peptide antibodies were located close to the C-terminus of hirudin, since the bond between Glu-61 and Glu-62 was not cleaved by the V8 staphylococcal protease in the presence of these antibodies. The epitope of the antibody 4049-83-12 was strictly conformation-dependent, it recognized neither S-carboxymethylated hirudin nor any peptides of hirudin. The cleavage of the bond between Glu-43 and Gly-44 by V8 protease, as well as the cleavage of the bond between Lys-47 and Pro-48 by lysyl endopeptidase, was prevented by the binding of the antibody 4049-83-12 to hirudin. The possibility that this epitope overlapped with a region of hirudin involved in the binding to thrombin is discussed.
Pharmaceuticals Research Laboratories, Ciba-Geigy Limited, Basel, Switzerland.
J M
JM
Schlaeppi
S
S
Vekemans
H
H
Rink
J Y
JY
Chang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D001666:Binding Sites, Antibody; D001780:Blood Coagulation Tests; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006629:Hirudins / Q000031:analogs & derivatives / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology; D012697:Serine Endopeptidases; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013917:Thrombin; C036813:lysyl endopeptidase; C060225:desirudin