Radioimmunoassay for for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin

Abstract

A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudinthrombin interaction, a monoclonal antibody to human α-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r. hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7×10 −3 to 2×10 −2 antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured α-thrombin (from 2×10 −4 to 1× l0 −2 NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I- r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1mg r.hirudin [Val 1-Val 2] into human volunteers. The plasma concentrations and elimination kinetics of r. hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.