Anti-hirudin Monoclonal Antibodies Directed toward Discontinuous Epitopes of the Hirudin Amino-Terminal and Epitopes Involving the Carboxy-Terminal Hirudin Amino-Acids

Abstract

A panel of eight monoclonal antibodies (MAbs) was obtained against recombinant hirudin variant 2 (rHV2). Specificities of the eight MAbs indicate that four of them recognize C-terminal amino acid residues (Group A) and four are directed against discontinuous epitopes and recognize a determinant (or determinants) within the 43 N-terminal residues (Group B). Using these antibodies recombinant hirudins missing one or more C-terminal amino acids can be distinguished from molecules with an intact C-terminus either in enzyme immunoassays (EIAs) or by immunoaffinity chromatography. A sandwich ETA using the combination of two antibodies, one from each group, can quantitate both recombinant hirudin variant 1 (rHV1) and rHV2 with a detection range from 1 to 10 ng/ml in either buffer or plasma. Using only one MAb a competitive antibody capture EIA can quantitate recombinant or natural hirudin variants 1, 2, and 3 with a detection range from 5 to 100 ng/ml for rHV2 with a lysine in position 47 (rHV2K47). None of the antibodies recognizes hirudin after it is complexed to α-thrombin. The ability of any one of these anti-rHV2 antibodies to interfere with hirudin binding to α-thrombin as measured by inhibition of thrombin′s amidolytic activity correlates with the range of MAb affinity constants (KD = 3.5 × 10−9 to 1 × 10−6 M). Incubating hirudin with one antibody from Group A (KD = 3.5 × 10−8M) and one from Group B (KD = 6.0 × 10−9 M) completely blocks the ability of hirudin to bind α-thrombin. This MAb panel is thus useful for probing the recombinant C-terminal integrity of hirudin, for sensitive free hirudin quantitations, and the combined use of two MAbs has potential applications as an antidote for hirudin in vivo.