Preparation of monoclonal antibodies to the thrombin/hirudin complex.

Monoclonal antibodies (mAbs) binding to the thrombin/hirudin (T.H) complex were prepared by either immunizing mice with hirudin and by screening for the mAbs cross-reacting with the T.H complex (group I), or by immunizing the animals directly with the T.H complex (group II). Epitope mapping of the mAbs of group I indicated that all the mAbs were binding to the hirudin N-terminal core domain only (residues 1-43). Among the mAbs raised against the T.H complex (group II), one mAb recognized an antigenic determinant expressed selectively upon binding of hirudin to thrombin. A double antibody sandwich type ELISA combining the mAb of group II with a mAb of group I was developed, allowing the determination of the T.H complex in human citrated plasma down to nanogram concentration levels.