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. Copyright © 1990, Wiley Blackwell. All rights reserved