Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site.

Hirudin is a 65 amino acid anticoagulant peptide produced in the leech. The single polypeptide is cross-linked by three disulfide linkages in the NH2 terminal half of the molecule. A peptide corresponding to the COOH terminus (residues 45-65) was synthesized utilizing lysine 47 as a specific residue to conjugate to thyroglobulin as a carrier for raising antibodies in mice. Using an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the major antigenic domain(s) was located between residues 52-65. The COOH terminal residues Ile-59, Tyr-63, and Leu-64 are crucial for maintaining the antigenic structure. The NH2 terminal region (residues 45-52) that is proximal to the carrier protein, however, was not immunoreactive. A possible mechanism by which antibodies recognize the COOH terminal region of the synthetic peptide and the strategy for raising such antibodies are discussed.