Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities.
Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.
Knoller
S
S
Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University, Israel.
Savion
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000990:Antithrombin III / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D005260:Female; D006493:Heparin / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000990:Antithrombin III; D006493:Heparin