Anti-hirudin monoclonal antibodies directed toward discontinuous epitopes of the hirudin amino-terminal and epitopes involving the carboxy-terminal hirudin amino acids.
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 EIA 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 alpha-thrombin. The ability of any one of these anti-rHV2 antibodies to interfere with hirudin binding to alpha-thrombin as measured by inhibition of thrombin's amidolytic activity correlates with the range of MAb affinity constants (KD = 3.5 x 10(-9) to 1 x 10(-6) M). Incubating hirudin with one antibody from Group A (KD = 3.5 x 10(-8) M) and one from Group B (KD = 6.0 x 10(-9) M) completely blocks the ability of hirudin to bind alpha-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.
Koch
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Hybridoma Laboratory, Statens Seruminstitut, Copenhagen, Denmark.
Whitechurch
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Cordier
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Roitsch
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004168:Diphtheria Toxoid; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D005260:Female; D014644:Genetic Variation; D006629:Hirudins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012441:Saccharomyces cerevisiae
D000911:Antibodies, Monoclonal; D004168:Diphtheria Toxoid; D000939:Epitopes; D006629:Hirudins; D011994:Recombinant Proteins